Variation in the Fine Structure of a Marine Achromobacter and a Marine Pseudomonad Grown Under Selected Nutritional and Temperature Regimes

Certain features of the fine structure of a marine achromobacter and a marine pseudomonad were dependent upon the conditions of growth. Cells of achromobacter grown at 10 C in a low peptone-seawater (SW) medium displayed the characteristic morphology of the achromobacter: a regularly undulant outer element of the cell wall and a planar inner element, tightly packed ribonucleoprotein (RNP) particles in the cytoplasm, deoxyribonucleic acid (DNA) disposed in a lobate manner, and dense inclusion bodies. Few mesosomes, however, were seen. Cells of achromobacter grown at 10 C in a high peptone-SW medium had larger and more highly organized mesosomes. At 22 C, in a low peptone-SW medium, no mesosomes were seen, but the inclusions were more frequently seen and were larger in the achromobacter cells. At 22 C, in a high peptone-SW medium, these cells revealed the greatest variation in cellular morphology. They contained both small and large mesosomes, or no mesosomes, and both small and large inclusions, or no inclusions. Pseudomonad cells at 10 C in a low peptone-SW medium revealed a typical gram-negative morphology: double-layered, irregularly undulant cell wall; more nearly planar cytoplasmic membrane; densely stained, lightly packed RNP particles; finely fibrillar, axially disposed DNA; simple mesosomes. At 10 C, in a high peptone-SW medium, pseudomonad cells revealed associated strands of material and intracytoplasmic ringlike structures. At 22 C, in a low peptone-SW medium, pseudomonad cells had a more undulant cell-wall and a more nearly planar cytoplasmic membrane. At 22 C, in a high peptone-SW medium, these cells revealed prominent blebs of the cell wall.     

Identity of Holmes Positive Bodies with Electron Microscopically Demonstrable Nucleolus-Like Bodies in Neuronal Cytoplasm

By light microscopic observation of mouse brain stained by Holmes' silver method deeply stained cytoplasmic inclusion bodies were seen in almost all nerve cells of the locus coeruleus. Electron microscopy of tissue samples from floating Vibratome sections stained by Holmes' silver method demonstrated that the nucleolus-like bodies in the cytoplasm were densely impregnated with gold particles. Hence, it was confirmed that the cytoplasmic inclusion bodies of paraffin sections stained by Holmes' method are identical to the so-called nucleolus-like bodies seen in electron microscopic studies.     

Electron Microscopic Observations on the Structure of the Envelopes of Mature Elementary Bodies and Developmental Reticulate Forms of Chlamydia psittaci

Purified suspensions of Chlamydia psittaci were prepared from L cells. Thin sections of intact elementary bodies and intact developmental reticulate bodies and of their purified envelopes were observed by electron microscopy. In both intact organisms and partially purified envelopes, two membranous structures, each appearing in electron micrographs as two darkly stained layers, were observed. In the elementary body sections, the outer membrane was round, apparently rigid, and was not soluble in 0.5% sodium dodecyl sulfate. The inner layer was irregular in shape and was completely removed by detergent treatment. We interpret these results to indicate that the outer rigid layer of the envelope is the cell wall and the inner layer is the cytoplasmic membrane. When the fragile reticulate body envelopes were similarly studied, the outer cell wall was clearly visible, and some evidence of an inner membrane was seen. After treatment with nucleases and detergent, all evidence of inner or cytoplasmic membrane was removed, but the outer cell wall remained. Thus, it appears that the cell wall of this organism is continuous throughout the growth cycle and that the fragility and lack of rigidity of the reticulate body cell is due to changes in chemical composition or structure of the cell wall.     

Unusual ultrastructural features in three strains of Cyanothece (cyanobacteria)

Three unicellular cyanobacterial strains (PCC 7425, PCC 8303, PCC 9308) assigned to the genus Cyanothece Komárek 1976, which showed an unusually high content of light refractile inclusions when viewed by phase-contrast microscopy, were characterized by confocal laser scanning microscopy and transmission electron microscopy. All strains had concentric cortical thylakoids and a compact central nucleoid. Frequently, the two innermost thylakoid membranes protruded to form circular enclosures containing cytoplasm or electron-transparent granules, or both. The largest granules were partially immersed in the nucleoid region, but they remained attached to the inner cortical thylakoids by a single narrow connection. The pattern of binary cell division in strain PCC 7425 was different than that in strains PCC 8303 and PCC 9308. In the former, all cell wall layers invaginated simultaneously, whereas in the latter the invagination of the outer membrane was delayed compared to that of the cytoplasmic membrane and the peptidoglycan layer. Thus, prior to completion of cell division, the new daughter cells of strains PCC 8303 and PCC 9308 were transiently connected by a thick septum, which was not observed in strain PCC 7425. Nucleoid partitioning coincided with initiation of cell division in all three strains and was unlike that reported in other bacteria and in archaea, in which separation of the nucleoids precedes cell division. Based on the common morphological and ultrastructural features, the three strains of Cyanothece examined constitute a distinct cluster, which might deserve independent generic status.     

Ultrastructure of L forms. IV. L forms of nonagglutinating vibrios]

A study was made of the ultrastructure of stable L-forms of Nag vibrios aged 24 hours. Cells of all types of the L-forms had cytoplasmic membranes, and a three-layered structure, which was found not everywhere. Externally of the cytoplasmic membrane, in some areas of the individual cells there were revealed a plastic layer of cell wall and a basal membrane. However, in difference to bacterial forms of the vibryos, rigidity of the cell wall was disturbed, and the links between the cell wall and the cytoplasmic membrane were indetectable. There were regularly revealed lamellar of myelin-like membranous structures in the cytoplasm, which did not occur in bacterial forms, and also lamellar mesosomes. The latter were found in the sites of cell division. Viability of small bodies as the minimal reproductive forms of the L-cultures is confirmed by the presence in them of a nucleoid and of the binary division.     

Freeze-Etch Study of Pseudomonas aeruginosa: Localization Within the Cell Wall of an Ethylenediaminetetraacetate-Extractable Component

A freeze-etch study of normal cells of Pseudomonas aeruginosa and of cells after incubation with ethylenediaminetetraacetate (EDTA) and tris(hydroxymethyl)aminomethane (Tris) was performed. When cells were freeze-etched without a cryoprotective agent, a smooth outer cell wall layer, which showed a regular array of subunits, and the presence of flagella and pili were observed. These features were not observed in cells freeze-etched after cryoprotection with glycerol. Four fracture surfaces, which resulted from splitting down the center of the outer wall membrane and of the inner cytoplasmic membrane, were revealed in freeze-etched glycerol-protected cells. The murein layer was seen in profile between the outer cell wall membrane and the cytoplasmic membrane. Spherical units and small rods composed of the spherical units were observed in the inner layer of the outer cell wall membrane. These spherical units appeared to be attached to, or embedded in, the inner face of the outer layer of the outer cell wall membrane. These spherical units were removed from cells on exposure to EDTA-Tris, resulting in cells that were osmotically fragile. The spherical units were detected via electron microscopy of negatively stained preparations in the supernatant fluid of cellular suspensions treated with EDTA-Tris. Upon addition of Mg(2+), the spherical units were reaggregated into the inner layer of the outer cell wall membrane and the cells were restored to osmotic stability. The spherical units were shown to consist primarily of protein. These data are thought to represent the first ultrastructural demonstration of reaggregation of cell wall components within a living cell system.     

Heliobacterium chlorum: cell organization and structure

The basic cellular organization of Heliobacterium chlorum is described using the freeze-etching technique. Internal cell membranes have not been observed in most cells, leading to the conclusion that the photosynthetic apparatus of these organisms must be localized in the cell membrane of the bacterium. The two fracture faces of the cell membrane are markedly different. The cytoplasmic (PF) face is covered with densely packed particles averaging 8 nm in diameter, while the exoplasmic (EF) face contains far fewer particles, averaging approximately 10 nm in diameter. Although a few differentiated regions were noted within these fracture faces, the overall appearance of the cell membrane was remarkably uniform. The Heliobacterium chlorum cell wall is a strikingly regular structure, composed of repeating subunits arranged in a rectangular pattern at a spacing of 11 nm in either direction. We have isolated cell wall fragments by brief sonication in distilled water, and visualized the cell wall structure by negative staining as well as deep-etching.Abbreviations PF protoplasmic fracture face - EF exoplasmic fracture face     

Ultrastructural changes in the spermatid nuclear envelope of Periplaneta americana during spermiogenesis

The nuclear envelope in the earliest stage of spermatid development possesses double membranes with pores aggregated in certain areas and arranged in a hexagonal pattern. The convex face of the outer nuclear membrane is relatively smooth and the convex face of the inner nuclear membrane carries many particles which are arranged in net-like pattern. In the later developmental stages, nuclear pores were not observed, the convex face of the inner nuclear membrane being covered with densely packed particles and the convex face of the outer nuclear membrane having a rough appearance. During the final stage of spermatid development, the cross-fractured face of the nucleus reveals the profiles of the nuclear fibres, the granular appearance of the convex face of the outer nuclear membrane, and the convex face of the inner nuclear membrane carrying more densely packed particles.     

Regular structures in membranes: the lumenal plasma membrane of the cow urinary bladder.

The ultrastructure of the lumenal plasma membrane of the cow urianry bladder has been studied in thin sections of glutaraldehyde- and glutaraldehyde-H2O2-fixed specimens, by negative staining and freeze fracture. A regular hexagonal array of particles confined to polygonal plaques 0-1-0-4-mum in diameter and separated by 0-02-mum interplaque areas is revealed by all 3 techniques. Cross-sections through particulate areas fixed with glutarayldehyde-H2O2 display a tetralaminar structure consisting of the usual approximately 8-nm-thick trilamellar unit membrane structure, on the external dense leaflet of which is located an additional approximately 4-nm-thick stratum which is occasionally resolved into a row of regulrly spaced approximately 4-nm-diameter particles. Non-particulate areas feature only the approximately 8-nm-thick trilamellar structure. Tangential sections reveal an hexagonal array of particles with a unit cell of approximately 16 nm. Four membrane faces can be revealed by freeze fracture and etching of membranes of the cow urinary bladder; 2 complementary split inner membrane faces (A and B) revealed by the cleaving process and the lumenal and cytoplasmic membrane surfaces exposed by etching. Face B, which belongs to the external membrane leaflet and faces the cytoplasm, displays plaques of particles arranged in a hexagonal lattice with a unit cell of approximately 16 nm. Face A, which belongs to the cytoplasmic membrane leaflet and faces the lumen, displays a complementary array of hexagonally packed pits. The hexagonally arranged particles also protrude into the lumenal membrane surface where they can occasionally be resolved into 6 approximately 5-nm-diameter subunits; the cytoplasmic surface appears smooth. Six approximately 5-nm-diameter subunits are also revealed in negatively stained preparations. The data are consistent with a model for the membrane in which the particles forming the hexagonal structure protrude above the lumenal membrane surface and also bridge most of the thickness of the membrane.     

Ultrastructure of the Membrane System in Lactobacillus plantarum

Electron microscopic study of Lactobacillus plantarum revealed mesosomes in different stages of maturation and structural relation with other cell organelles. Small, immature mesosomes were bounded by a prominent electron-dense layer with another extremely faint layer on the outside. This corresponds to the appearance of the cytoplasmic membrane. Large mature mesosomes were surrounded by a triple-layered unit membrane having electron-opaque layers of approximately equal density, suggesting that the composition of the boundary membrane alters during development of this structure. Three-dimensional observations derived from serial sections indicated that mesosomes always maintain a connection between the cytoplasmic membrane and the comparable layers of their boundary. The cytoplasmic membrane also consisted of a triple-layered unit membrane, the innermost layer of which was less electron-opaque and was usually hidden by the relatively dense background of the cytoplasm. The innermost layer of the cytoplasmic membrane was most clearly seen in plasmolyzed cells. Only mature mesosomes made distinct contacts with, or were partially immersed in, the nucleoplasm. The boundary of such mesosomes frequently seemed to be discontinuous, suggesting that the mesosome interior was in direct contact with the nucleoplasm. Mesosomes involved in cross-wall formation at a division plane increased in size and passed through a sequence of positions which led ultimately to an association with the nucleoplasms of the daughter cells. The inner surface of the cell wall was lined by a thin, electron-dense layer whose composition and function are unknown. Under the cultural conditions used, this organism regularly contained a polyphosphate granule.     

Biguanide-induced changes in Acanthamoeba castellanii: an electron microscopic study

Trophozoites and cysts of Acanthamoeba castellanii were exposed to chlorhexidine diacetate (CHA) and polyhexamethylene biguanide (PHMB); changes in cell ultrastructure and surface structure were examined by both transmission and scanning electron microscopy. PHMB caused a greater degree of structural and membrane damage; the cytoplasmic contents were severely depleted and there were clusters of densely stained precipitates on the cell surface. Concentrations of CHA greater than 100 μg ml⁻¹ produced shrinkage from the cyst wall. At high concentrations, PHMB induced a slight withdrawal of the cytoplasm from the wall and, unlike CHA, induced swelling of the cysts. These findings do not define the mechanisms of action of CHA and PHMB, but provide evidence that a major target site for both agents is the plasma membrane. However, additional intracellular damage undoubtedly contributes to the lethal effects. The greater resistance of cysts may be associated with reduced biguanide uptake.     

Biguanide-induced changes in Acanthamoeba castellanii: an electron microscopic study

Trophozoites and cysts of Acanthamoeba castellanii were exposed to chlorhexidine diacetate (CHA) and polyhexamethylene biguanide (PHMB); changes in cell ultrastructure and surface structure were examined by both transmission and scanning electron microscopy. PHMB caused a greater degree of structural and membrane damage; the cytoplasmic contents were severely depleted and there were clusters of densely stained precipitates on the cell surface. Concentrations of CHA greater than 100 microg ml(-1) produced shrinkage from the cyst wall. At high concentrations, PHMB induced a slight withdrawal of the cytoplasm from the wall and, unlike CHA, induced swelling of the cysts. These findings do not define the mechanisms of action of CHA and PHMB, but provide evidence that a major target site for both agents is the plasma membrane. However, additional intracellular damage undoubtedly contributes to the lethal effects. The greater resistance of cysts may be associated with reduced biguanide uptake.     

Fine Structure of Cryptococcus neoformans Grown In Vitro as Observed by Freeze-Etching

Cryptococcus neoformans grown on culture media was observed by the freeze-etching technique. In the capsule, short fibrils were seen when freezeetched. This organism was unique in the appearance of the cell wall, which showed two strata. The outer one was dense with particles of about 20 nm in diameter, whereas the inner one was sparse in particles. The appearance of the cell membrane of this organism differed distinctly depending on the culture media. When grown on glycerol medium, the cell membrane possessed, as do other yeasts, clear but somewhat longer and curved invaginations. The membrane of cells grown on nonglycerol medium exhibited, however, only a few invaginations of irregular shape. Instead, characteristically of this organism, the cell membrane showed round depressions of 40 to 200 nm in diameter which were the surface view of the paramural bodies. In cross-fractured cells, both types of paramural bodies were found. Some of them contained a single vesicle of about 50 nm in diameter. These seem to play a role in secreting the cytoplasmic vesicles. Data suggesting the existence of multivesicular bodies in the cytoplasm and multivesicular lomasomes were also obtained. Some of the baglike paramural bodies showed multilayered membrane. These are thought to be plasmalemmasomes. This organism was similar to other yeasts reported in other respects.     

STRUCTURAL FEATURES OF MESOSOMES (CHONDRIOIDS) OF BACILLUS SUBTILIS AFTER FREEZE-ETCHING

Freeze-etched cells of Bacillus subtilis have been studied with the electron microscope. The outer surface of the plasma membrane, i.e. the side facing the cell wall, is covered with numerous granules and short strands, each measuring approximately 50 A in diameter. These strands are occasionally seen to enter the cell wall. The inner surface of the plasma membrane, i.e. the side facing the cytoplasm, appears to be sparsely dotted with small particles measuring about 50 A. The envelope of mesosomes differs from the plasma membrane. Blunt protrusions arise from its outer surface; the inner surface appears smooth. Stalked particles, as described by other investigators after negative staining with phosphotungstic acid, were not observed on any membrane surface in our material. Preparations were also made of specimens prefixed in osmium tetroxide prior to freeze-etching. Under these conditions the bacterial membranes appeared to be surprisingly well preserved. In contrast to directly frozen, unfixed cells, some osmium tetroxide-fixed preparations showed a differentiation in cytoplasm and nucleoplasm, which made it possible to observe the close association of the mesosome with the latter.     

DEVELOPMENT OF THE EMBRYONIC CHICKEN THYMUS III. UNEQUAL DIVISION OF LYMPHOID CELLS

Cell division of thymus lymphoid cells from 11- to 17-day old embryonic chickens, as well as chickens just after hatch was investigated on cell smears stained with Giemsa. Unequally dividing cells were observed in the developmental stage of thymocytes. At the telophase of such cells, the cytoplasm of one of two future daughter cells was apparently larger in amount and was sometimes stained deeper than the cytoplasm of its counterpart. Unequal division was also observed in pro-, meta- and anaphase; sometimes a dividing cell had a large cytoplasmic process belonging to one hemisphere, suggesting that only one of the two daughter cells would receive the cytoplasmic process through cell division.
The incidence of unequal division calculated by a rough estimation was around 10% of the total cell division between 11 and 13 days of embryonic development, and decreased progressively thereafter.     

Fine Structure of Strictly Anaerobic,Non-Spore-Forming,Gram-Negative Bacilli

The fine structure of a strain of Bacteroides insolitus has been studied by ultrathin sectioning and electron microscopy. Logarithmically growing cells were fixed both by osmic acid and potassium permanganate, and embedded in Epon. Thin sections were stained with uranyl acetate and examined. The periphery of the cell was composed of a wavy three-layered outer membrane (ca. 80 A), an intermediate layer (50–200 A), and three layered cytoplasmic membrane (ca. 80 A). Single or double bridges which connected the outer membrane with the cytoplasmic membrane were observed. Invagination of the cytoplasmic membrane was observed in no occasion. Independent, distinct, and uniform particles were not the main component of the cytoplasm. The cytoplasm was filled with more or less beaded reticulum-like structures. The nucleoplasm with fine fibrils was mainly dispersed continuously in rather regular cubic masses in an intermediate region between the center and the periphery of the cell. Contacts of the nucleoplasm with the cytoplasmic membrane were occasionally observed.     

Fine Structure of Bacillus megaterium during Microcycle Sporogenesis

Ultrathin sections were prepared from cultures of Bacillus megaterium QM B1551 undergoing microcycle sporogenesis (initial spore to primary cell to second-stage spore without intervening cell division) on a chemically defined medium. The cytoplasmic core of the dormant spore was surrounded by plasma membrane, cell-wall primordium, cortex, outer cortical layer, and spore coats. Early in the cycle, the coat opened at the germinal groove, the cortex swelled, ribosomes and a chromatinic area associated with large mesosomes (which may later be incorporated into the expanding plasma membrane) appeared in the core, and the cell wall became defined at the site of the cell wall primordium. Poly-β-hydroxybutyrate granules began to appear in the primary cell at about 3 hr. By 7 hr, the forespore of the second-stage spore was delineated by typical double membranes. Between 7 and 12 hr, second-stage cell-wall primordium and cortex developed between the separating forespore membranes. The inner membrane became the plasma membrane of the second-stage spore, and the outer membrane eventually disintegrated within the second-stage spore cortex. A densely staining double layer (spore-coat primordium) developed external to the outer forespore membrane. The inner spore coat and the outer cortical layer of the second-stage spore developed from this primordium. The outer part of the spore coat, probably of sporangial origin, was laid down on the external surface of the inner spore coat. By 12 hr, second-stage spores were almost mature. By 20 hr, the mature endospores, with a thickened outer coat, were often still enclosed by degenerate primary cell wall and by the outer cortical layer and spore coat of the initial spore.     

Early death at medium acidification and survival after low pH adaptation inCryptococcus neoformans

WhenCryptococcus neoformans was grown in yeast nitrogen base (YNB) supplemented with 0.5% glucose, the medium was acidified to below pH 3 during the exponential growth phase, which caused early growth-phase death in susceptible strains. Even in resistant strains, 30–70% cells died if incubated for 2 d in YNB supplemented with 1.5% glucose, whereas the remaining cells survived long. Two types of fatal alterations have been observed in dead cells. In the first type, release of cytoplasm occurred through weakened parts of the cell wall; structures attached to cell walls of dead cells were shown to be rich in proteins by FITC staining, indicating their cytoplasmic origin. In the second type, cells shrank distinctly with no sign of wall rupture. The shrinkage may be due to dysfunction of the plasma membrane at low pH. The mechanism of cell survival in medium below pH 3 was also examined. Aniline blue alone, or calcofluor together with methylene blue, allowed cell wall glucan or chitin and dead cell cytoplasm to be stained simultaneously. In the later stages of incubation, cells showing bright staining for cell wall glucan and chitin emerged. These changes in cell wall synthesis could be considered as an adaptation mechanism to acidification of the medium, because such cells survived longer than cells showing no change in the cell wall staining pattern.     

Fine Structure of Listeria monocytogenes in Relation to Protoplast Formation

Among the eight strains of Listeria monocytogenes tested for lysozyme sensitivity, two were resistant to lysozyme but became sensitive after lipase pretreatment. Among the other six, one was very sensitive to lipase and another one was extremely susceptible to lysozyme. Stable protoplasts were formed from the lysozyme-resistant strain (42) by lipase and lysozyme treatment, which completely digested the cell wall. The cell wall (uranyl acetate-lead stained) was of a thick triple-layered profile, with the intermediate layer of low density. Lipase treatment for a short time (60 min) did not cause any alteration in structure, but prolonged treatment (180 min) caused extensive digestion of the plasma membrane and the cell wall, liberating cytoplasmic material. When the cells were treated with either lipase or lysozyme, a small number of protoplasts were extruded through the partly digested or weakened transverse cell wall, leaving an almost intact cell wall ghost. There were small vesicular structures in the interspace between cell wall and plasma membrane. Mesosomes of varied organization were prominent in electron micrographs, both in sections and in negatively stained preparations. These were largely everted during protoplasting in the form of tubules and as small peripheral buds; a few small vesicles also remained as intrusive structures, some of which were very unusual because they appeared to be enclosed by the inner layer of plasma membrane alone. Lysis of the protoplasts by dilution of the sucrose, while maintaining a constant ionic environment, liberated many small vesicular structures and fibrillar nuclear material.     

In situ characterization of Clostridium botulinum neurotoxin synthesis and export

A monoclonal antitoxin/colloidal gold probe and sequential centrifugation were used to study synthesis, translocation and export of Clostridium botulinum strain 62A neurotoxin (NT). Exponential growth occurred after 5 h of anaerobic incubation of spores and continued for 15–16 h. NT was detected at 15 h using the probe and transmission electron microscopy (TEM), 2 h earlier than the first detection by the mouse bioassay. During exponential growth, the probe localized NT primarily in the cytoplasm, on the inner side of the cytoplasmic membrane and in the cell wall. During stationary and death phases, the NT was located within the cytoplasm, cell wall and extracellularly. NT was released from the cell during cell wall exfoliation. Cells retained NT after repeated gelatin-phosphate washes and sequential centrifugations, consistent with the TEM observation that the NT is bound to the cell wall. These observations indicate that the process of Cl. botulinum type A NT production follows a sequence of synthesis, translocation across the cytoplasmic membrane and export through the cell wall.