Morphological Observations of Diplodia maydis on Synthetic and Natural Substrates as Revealed by Scanning Electron Microscopy

Mycelial and spore morphology of Diplodia maydis were investigated by using scanning electron microscopy after growth on various media and natural substrates (oat and corn kernels, and corn husks). Of several specimen preparation methods studied, Parducz fixation followed by critical-point or freeze-drying gave adequate preservation for pycnidia, mycelia, and spores. Morphological characteristics were similar in rotary and reciprocal shaker cultures and differed from that found in stationary cultures in the amount of slime-like material produced and precipitated matter on the mycelial surfaces. In general, mycelial surfaces were smooth. Large areas of coalesced material were present in all samples examined. Slime-like material produced in liquid media appeared as a finely laced net, randomly appearing throughout the mycelia with bead-like structures present along the net. A fine netting also was observed interspersed among the spores inside the pycnidia obtained from oats. Slime-like material was observed to cover the pycnidia produced on oat and corn kernels. In the latter case, the spores were less protected by the outer slime-like covering. Thickened node-like structures were observed in mycelial mats produced in modified Fries 2 medium, on potato dextrose agar plates, and on infected oats. Round and ovate thickened node-like structures were observed in mycelium produced on corn kernels. In general, node-like structures were less abundant in mycelia from naturally infected substrates. Conidia were commonly rounded to tapered and two celled, with a distinctive ridged septum at the middle. Dried spores were collapsed in a characteristic flask-like fashion.     

Fertilization Cone of Carp Eggs as Revealed by Scanning Electron Microscopy

The process of formation of the fertilization cone in carp eggs was examined by scanning electron microscopy. The fertilized eggs responded to penetration of one sperm by primary and secondary steps of formation of a fertilization cone of unique morphology. In the primary step, the earliest fertilization cone was seen at the superior or anterosuperior part of a fused sperm head in inseminated eggs fixed 20 sec after immersion in fresh water. The cone reached a maximum of more than 10 μm in length and 3–4 μm in thickness by 40 sec, resulting in a transient plugging of the micropylar canal. In the secondary step, usually seen at 105–120 sec, a conformation reminiscent of a very small caldera volcano was formed, with the shortened earlier cone and part of the sperm tail at its top. By 2.5 min, the fertilization cone had become conical, and the sperm tail still extended from its top. At 3 min, the sperm tail was often not detectable, but a cytoplasmic eminence was still seen as a trace of the fertilization cone. The role of the earlier fertilization cone in blocking polyspermy is discussed.     

Structure of Trichomonads as Revealed by Scanning Electron Microscopy*†

SYNOPSIS.
A scanning electron microscope (SEM) study of Hypotrichomonas acosta (Moskowitz), Trichomonas vaginalis Donné. Pentatrichomonas hominis (Davaine), and Tritrichomonas foetus (Riedmüller) provided new information about the structure of the periflagellar canal: emergence of the flagella from the cell body; structure of the undulating membrane; and position, shape, and size of the pelta. Of special interest were the spatial relationships of the attached part of the recurrent flagellum and the accessory filament in Hypotrichomonas and in the members of Trichomonadinae, i.e. Trichomonas and Pentatrichomonas.     

Accumulation and Distribution of Extracellular Matrix as Revealed by Scanning Electron Microscopy in Freeze-Dried Newt Embryos Before and During Gastrulation

A scanning electron-microscopic study was carried out on the extracellular matrices (ECMs) in freeze-dried newt embryos from the cleavage to the gastrula stage. The results revealed the appearance, accumulation and distribution of two types of ECMs, a fibrillar ECM in the blastocoel and an amorphous ECM on the inner surface of the blastocoelic wall (BW). The fibrillar ECM first appeared in the blastocoel at the cleavage stage and increased notably in quantity at the blastula and gastrula stages. On the other hand, the amorphous ECM was initially detected on the inner surface of the BW at the beginning of gastrulation and it increased in quantity during gastrulation. With the progress of archenteron invagination, the amorphous ECM was found to be deposited in the space between the BW and migrating cells.     

Surface Features of Bacillus polymyxa Spores as Revealed by Scanning Electron Microscopy

The surface features of Bacillus polymyxa spores were compared by use of thin sections, carbon replicas, and the scanning electron microscope. Some features of the characteristic ridges, previously reported in ultrathin sections and carbon replicas of spores of this species, were more clearly revealed with the scanning electron microscope. A three-dimensional image is provided because of the greater depth of focus possible with this instrument. End-on views of B. polymyxa spores readily illustrate the polygonal porelike structure present.     

Mode of Infection of Phyllosticta maculata on Banana as Revealed by Scanning Electron Microscopy

The initial infection stages of Phyllosticta maculata on banana were studied using scanning electron microscopy. Conidial germination on the banana leaf surface commenced within 3 h postinoculation to produce a long and slender germ tube. The hyphae developed secondary branches and mostly grew randomly across the leaf surface. Appressoria were formed at the apex of the germ tubes within 18 h postinoculation and were variable in shape. A layer of an extracellular matrix surrounded the appressoria at the pathogen–host interface. On the fruit surface, conidia germinated to produce predominantly swollen germ tubes which functioned as lateral appressoria together with some slender ones. These germ tubes were formed within 3 h postinoculation. There was no stomatal penetration apparent on the leaf; instead, direct penetration through the cuticle with and without the formation of appressoria was observed. Cuticular degradation on the leaf surface was evident with a circular, darkened area around the point of penetration by hyphae or appressoria. The significant role of pycnidia and conidia in the epidemiology of the disease was further demonstrated in naturally infected leaf samples.     

Structure of Monocercomonas sp. as Revealed by Electron Microscopy*

SYNOPSIS. Monocercomonas shares many fine-structural features with all other trichomonads. These include the basic arrangement of the kinetosomes as well as of the recurrent and 3 anterior flagella. The pelta-axostyle complex and the parabasal apparatus, i.e. the Golgi complex and the periodic filaments, also conform to the trichomonad pattern. Of interest with regard to the crucial evolutionary position of Monocercomonas, considered to represent the most primitive trichomonad type, is the fact that it has some structures in common with other Monocercomonadidae and Trichomonadinae and others in common with Devescovinidae and Tritrichomonadinae. Among the former organelles are the marginal lamella and the costal base, and among the latter, the comb-like organelle situated between the infrakinetosomal body and parabasal filament 2 as well as the infrakinetosomal body. No traces of either costa or undulating membrane have been noted, but a complex structure homologous to the marginal lamella of Hypotrichomonas and Trichomonadinae is found underlying the short anteriormost portion of the recurrent flagellum that is attached to the body surface. Observations of sections of selected division stages indicate the potential of parental kinetosomes #1 and #3 to become daughter kinetosome #2.     

An Internal View of the Spherical Body of Treponema macrodentium as Revealed by Scanning Electron Microscopy

The osmium-dimethyl sulfoxide-osmium method for clear visualization of intracellular structure was used to observe the detailed inner structure of the spherical bodies produced in vitro by a human oral treponeme. Scanning electron microscopy of the cracked spherical body revealed no morphological differences between the outer and inner surfaces of the spherical body membrane, and that multiple folded or somewhat linear main bodies adhere closely to the inner surface. In addition, axial flagella partially free from the main bodies spread widely within the body to make a network, and a number of blebs ranging from approximately 1 μm to 0.2 μm in diameter were located near the terminal or subterminal areas of the main bodies. The origin of the blebs and the mechanism of spherical body formation are discussed.     

Ultrastructure of Cryptosporidium parvum Oocysts and Excysting Sporozoites as Revealed by High Resolution Scanning Electron Microscopy1

Cryptosporidium parvum oocysts isolated from calf feces were examined by scanning electron microscopy during excystation. Intact C. parvum oocysts were spheroid to ellipsoid, ?3.5 × 4.0 μm, with length: width ratio = 1.17. The oocyst wall had a single suture at one pole, which spanned 1/3 to 1/2 the circumference of the oocyst. During excystation the suture dissolved, resulting in a slit-like opening, which the sporozoites used to exit the oocyst. Sporozoites were 3.8 times 0.6 μm and had a rough outer surface.     

Concanavalin A-Induced Morphological Changes and Its Binding Sites in Starfish Follicle Cells as Revealed by Electron Microscopy

Concanavalin A (Con A) stimulates the production in starfish follicle cells of 1-methyladenine, a hormone which induces oocyte maturation. We have therefore investigated Con A-induced morphological changes and Con A-binding sites in the follicle cell using native Con A and horseradish peroxidase- or ferritin-labeled Con A (HRP-Con A, Fer-Con A). After isolated follicle cells were incubated with Con A (1 mg/ml), vacuoles, the Golgi complex and multivesicular body-like organelles (MVBs) became prominent in most of the cells. After follicle cells were prefixed and then incubated with Fer-Con A for 60 min, tagged ferritin was diffusely and randomly distributed as single or small clustered particles on the cell surface. The incubation of isolated follicle cells with Fer-Con A for 10 min before fixation resulted in numerous ferritin particles localized along the internalized membrane, and also in vacuoles, MVBs and small lysosome-like structures. After 60 min incubation with Fer-Con A, ferritin was further located in large lysosome-like structures and in vesicles near and in the Golgi area as well as in the organelles described above. HRP-Con A binding sites were also observed in vacuoles and MVBs of the intact cells.
These results suggest that Con A binds at first to the cell surface and causes rapid internalization and that membrane-bound Con A is easily endocytosed into vacuoles, MVBs and lysosome-like structures, and is later incorporated in some vesicles in the Golgi area.     

Expression of Polarity during early Development of Microspore-derived and Zygotic Embryos of Brassica napus L. cv. Topas

Microspore derived (MS-)embryogenesis and zygotic embryogenesis of Brassica napus L. cv. Topas were investigated by light and scanning electron microscopy to reveal the expression of polarity during the transition phase from globular to heart and torpedo shape. During the first 5 days of MS-embryo formation, the cell wall of the former microspores remained intact and a globular mass of cells developed within. Pollen walls ruptured after 5 days of culture; embryos proceeded through heart-shape and torpedo-shape stages within 15 days in a way comparable to, but faster than observed during zygotic embryogenesis. Expression of polarity in globular and elongating MS-embryos was analyzed by detection of the distribution of activated calmodulin as well as of free cytosolic calcium by using confocal scanning laser microscopy, and by the detection of starch. Calmodulin was evenly distributed in globular embryos and only exhibited clear polar distribution in elongated embryos. Free cytosolic Ca²⁺ accumulated in the protoderm of globular embryos and in the central cylinder of torpedo shaped embryos, but never showed polar distribution. Accumulation of starch granules at the root poles of both sexual as well as MS-embryos, however, indicated polar distribution before the transition from globular to heart shape stage. Since the local rupture of the pollen wall of 6-day-old MS-embryos was never preceded by the decrease of starch at that site, it is likely that the rupture of the pollen wall plays an important role in the local activation of cell metabolism and thus in the determination of the polarity axis in MS-embryos.     

Transmission Electron Microscopy of Tissue Prepared for Scanning Electron Microscopy by Ethanol-Cryofracturing

Tissue processed for scanning electron microscopy by ethanol-cryofracturing combined with critical point drying was embedded and sectioned for transmission electron microscopy. Study of sections cut in a plane passing through the fracture edge indicated that preservation of cellular fine structure of fractured cells was excellent. Even at the most peripheral edge of the fracture there was no evidence that movement of cytoplasmic components occurred to distort the original structural organization of fractured cells. Lack of cytoplasmic detail in ethanol-cryofractographs has been due more to the nature of the fracturing of the tissue and to the obscuring effects of the metal coating than to structural deformation at the fracture edge or to limitations in resolving power of the scanning electron microscope used.     

Ultrastructure of the Squid Axon Membrane as Revealed by Freeze-Fracture Electron Microscopy

The structure of the axolemma of the squid giant axon was studied by freeze-fracture electron microscopy. Three types of preparations were examined: intact axons, axons with their Schwann cell sheaths stripped off prior to freezing, and axons with their Schwann cell sheaths chemically detached but not mechanically removed. Because of a problem of cross-fracturing, the first two types of preparations revealed very few membrane faces of the axolemma. This cross-fracturing problem, however, was eliminated when we used a complementary replication method to fracture the third type of preparation. We found that the E-face of the axon membrane was smooth relative to the P-face, which showed many prominent intramembrane particles (IMP). The diameters of the typical IMP range from 6 to 15 nm. The P-face of the adjacent Schwann cells also showed many large IMP. The sizes and heights of the Schwann-cell IMP, however, appear to be more homogeneous than the P-face axolemma.     

The Dimension of Trichomonas vaginalis as Measured by Scanning Electron Microscopy

It is known that physicochemical conditions (e.g., pH, temperature, and ionic strength) affect the size of trichomonads. In this study, the sizes of 4 isolates of Trichomonas vaginalis cultured for more than a year (called "old T") and 3 isolates freshly isolated from vaginitis cases (called "fresh T") were compared by scanning electron microscopy. Although the fresh T had shorter body length, body width, and flagellar length than old T, total length (about 26 µm), including body length, flagella length, and axostyle length was almost the same in the 2 groups. A striking difference was observed between the axostyles of the 2 groups; the axostyle length of the fresh T (8.2 µm) was more than twice as long as that of the old T (4.0 µm). However, in several parasitology textbooks, the length of T. vaginalis is said to vary widely from 7 to 32 µm, and its undulating membrane is said to extend about half way (53.5%) to the posterior end of the body. On the other hand, in our study, the undulating membrane was observed to extend more than 3/4 of the body length (72.1%) in old T, whereas in fresh T it could not be measured. Taken together, we suggest that T. vaginalis averages 26 (21-32) µm in total length, with 9.5 (7.4-11.4) µm of body length and 6.8 (5.3-7.7) µm of width, and its undulating membrane extending 3/4 of its body length. Therefore, these findings may provide useful information for morphological characteristics of T. vaginalis.     

Structure of Pentatrichomonas hominis (Davaine) as Revealed by Electron Microscopy*

SYNOPSIS. Fine structure of Pentatrichomonas hominis is described in the light of previous light microscopic findings. The relationships among kinetosomes #1-#4 and R are like those previously reported orhomonas gallinae, and the same is true of the rootlet filaments associated with the several kinetosomes. The kinetosome (I) of the independent flagellum is situated just behind the reflection of the sigmoid filaments of kinetosome #2 onto the pelta and parallels these filaments for a considerable distance. The peltaraxostylar junction consists of 3 layers: the capitulum of the axostyle (outer, the pelta (intermediate, and the sigmoid rootlets of kineto some #2 (inner). The pelta overlaps the axostylar capitulum to a variable extent. The parabasal body consists of elongate and flattened cisternae of smooth endoplasmic reticulum surrounded by numerous small vesicles. There are 2 typically cross-striated parabasal filaments, filament 2 probably contributing most, if not all, the material to the slender, periodic organelle that underlies the parabasal body and usually does not extend far beyond the posterior end of the nucleus. The periodic costa is paralleled by paracostal granules, but there are few, if any, paraxostylar granules. The ultrastructure of the costa appears to be a network of flattened hexagons, with a single fibril projecting thru each of the hexagonal areas. The major cross-striations are made up largely of densely-stained filaments which are occasionally cut in cross section. The undulating membrane consists of a cytoplasmic fold extending from the dorsal surface of the organism and of the attached part of the recurrent flagellum, which is closely applied to the fold. The segment of the membrane dorsal to the flagellum, presumably the “accessory filament,” contains the marginal lamella, a membrane folded upon itself and with periodicity virtually indistinguishable from that of the rootlet filament of kinetosome #1.     

Compartmentalization of Pancreatic Secretory Zymogen Granules as Revealed by Low-Voltage Transmission Electron Microscopy

Low-voltage (5-kV) transmission electron microscopy revealed a novel aspect of the pancreatic acinar cell secretory granules not previously detected by conventional (80-kV) transmission electron microscopy. Examination of ultra-thin (30-nm) sections of non-osmicated, stain-free pancreatic tissue sections by low-voltage electron microscopy revealed the existence of granules with non-homogeneous matrix and sub-compartments having circular or oval profiles of different electron densities and sizes. Such partition is completely masked when observing tissues after postfixation with osmium tetroxide by low-voltage transmission electron microscopy at 5 kV and/or when thicker sections (70 nm) are examined at 80 kV. This morphological partition reflects an internal compartmentalization of the granule content that was previously predicted by morphological, physiological, and biochemical means. It corresponds to the segregation of the different secretory proteins inside the granule as demonstrated by high-resolution immunocytochemistry and reflects a well-organized aggregation of the secretory proteins at the time of granule formation in the trans-Golgi. Such partition of the granule matrix undergoes changes under experimental conditions known to alter the secretory process such as stimulation of secretion or diabetes.     

Inclusion Bodies in Xenosomes Purified on Percoll Gradients as Revealed by Electron Microscopy

A method is described for the isolation and purification of xenosomes, intracytoplasmic bacterial symbionts of the marine hymenostome Parauronema acutum , using percoll gradients. Xenosomes isolated by this procedure retained both their ability to kill susceptible Uronema strains and to infect homologous and heterologous P. acutum strains. Unexpectedly, both killer and non-killer xenosomes were found to contain inclusion bodies, heretofore unseen in fixed whole cell preparations, in the form of double helices, which we have termed H-bodies. The nature and function of these bodies is unknown.     

Apoptosis and Morphology in Mouse Embryos by Confocal Laser Scanning Microscopy

Confocal laser scanning microscopy combined with a vital stain was used to study apoptosis in organogenesis-stage mouse embryos. Apoptosis has previously been visualized in whole embryos using the vital dyes acridine orange, Nile blue sulfate, and neutral red. In the present study, mouse embryos were harvested on Gestation Day 9 and stained with the vital lysosomal dye LysoTracker Red. Following incubation in the stain, embryos were fixed overnight in 4% paraformaldehyde, dehydrated in a graded methanol series, and cleared in benzyl alcohol/benzyl benzoate. The resulting embryo is almost transparent and retains specific LysoTracker Red staining. To achieve optical sectioning through embryos, it was necessary to use low-power objectives. With this procedure, the entire embryo can be optically sectioned and reconstructed in three dimensions to reveal areas of dye staining. Our results demonstrate specific regions undergoing programmed cell death in normal development and increased LysoTracker staining in embryos exposed to hydroxyurea. This procedure allows for the optical imaging of whole Day 9 ( approximately 22 somites) embryos that were greater than 700 microm thick in the z axis and can be applied to studies involving neural tube formation or other aspects of organogenesis.     

Response ofBrassica napus L. Microspore-derived embryos to exogenous abscisic acid and desiccation

Summary Microspore-derived embryos fromBrassica napus cv. Topas (low erucic acid) and Reston (high erucic acid) were subjected to treatment with abscisic acid (ABA) during late-stage embryo development and then dried under controlled relative humidities to mature dry seed levels of moisture. Exogenously medium-supplied ABA arrested growth and development, reduced moisture content, increased total fatty acids on a dry weight basis, and stimulated systhesis of proteins in microspore-derived embryos. ABA also resulted in a higher proportion of 22∶1 in cv. Reston (high 22∶1) and increased the level of fatty acid unsaturation in cv. Topas (low 22∶1). The accumulation of two proteins that co-migrated with cruciferin and napin on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gels were also promoted by exposure to ABA, and the degree of accumulation was dependent on the concentration and time of application of ABA. Controlled desiccation of microspore embryos, used to simulate normal maturation and dehydration of zygotic embryos during seed development, did not seem to cause an increase of either storage proteins, total fatty acids, or 22∶1 (in cv. Reston), suggesting that dehydration is not a prerequisite for these processes, at least in culturedBrassica embryos.