ULTRASTRUCTURAL LOCALIZATION OF MINERAL MATTER IN BACTERIAL SPORES BY MICROINCINERATION

The fine localization of mineral matter in spores of Bacillus megaterium and Bacillus cereus was studied by the technique of microincineration adapted for use with the electron microscope. The specimens, which included intact and thin-sectioned spores as well as shed spore coats, were burned either in the conventional way at high temperature or by a new technique using electrically excited oxygen at nearly room temperature. The ash residues were examined by bright field, dark field, and diffraction in the electron microscope and also with the phase contrast microscope. In some cases, the specimen was previewed in both microscopes before incineration. The results do not support a previous report that the mineral elements of the spore are confined to a peripheral layer, but rather indicate that the spore core as well as the coat are mineral-rich. The cortex may be deficient in minerals, but the possibility of artifact prevents a clear decision on this point. Incinerated B. megaterium spores show a highly ordered fine structure displaying 100 A periodicity in the ash of the middle layer of the coat. The nature of this structure is discussed, as is the technique which demonstrated it. The fine definition of the ash patterns, particularly those obtained with the low-temperature, excited-oxygen technique, suggests that microincineration may be generally useful in the study of fine structure.     

中国产异蒴藓属（Lyellia）植物形态解剖比较观察

利用扫描电镜法、徒手切片法对中国产的2种异蒴藓进行观察分析。分别观察了孢子大小、颜色、叶片和茎的长度和宽度等。结果表明异蒴藓属植物叶片的一些形态学和解剖学性状表现出了金发藓科植物所共有的特征,异蒴藓属在金发藓科中气孔较明显属于进化性状,而孢壁纹饰则属于原始性状。     

An Effective Method of Preparing Sections of Bacillus polymyxa Sporangia and Spores for Electron Microscopy

Bacillus polymyxa sporangia and spores were prepared for examination in the electron microscope by methods whose critical features were apparently: judicious use of vacuum, to encourage complete penetration of the embedding medium; the use of epoxy resins as embedding media; and cutting of the thin sections with a diamond knife. Electron micrographs of material prepared in this manner exhibit undeformed sporangial sections. Some of the structures revealed have been shown before, though perhaps less distinctly; other structures are revealed here for the first time. While this single study does not pretend to elucidate all the complexities of sporulation in bacteria, these and similar images should make this possible, and some mention of the preparatory techniques that lead to them seems advisable at this time.     

ELECTRON MICROSCOPY OF SPORES OF BACILLUS MEGATERIUM WITH SPECIAL REFERENCE TO THE EFFECTS OF FIXATION AND THIN SECTIONING

Resting spores of Bacillus megaterium appear uniformly opaque and undifferentiated under the electron microscope. Germinated spores and spores which have lost their dipicolinic acid underwent characteristic changes in structure. Spores fixed with KMnO₄ lose their dipicolinic acid. Spores fixed with OsO₄ under certain conditions retain their dipicolinic acid. When conventional sectioning procedures are used with either method of fixation, abnormal spore structure is produced as a result of the solution of cellular constitutents. Dry sections of unfixed spores embedded in methacrylate reveal the spore structure in a more normal state. Indirect evidence has been obtained for the existence of a penetration barrier at or near the outer edge of the cortex.     

Electron microscopy of the surfaces of bacillus spores

The surface structures of the spores of Bacillus cereus, Bacillus thuringiensis, and Brevibacillus laterosporus were studied by transmission and scanning electron microscopy. Platinum deposition and negative staining with uranyl acetate revealed appendages and exosporium in B. thuringiensis and B. cereus. The exosporium structure was visualized by negative staining and ultrathin sectioning. For staining the exosporium polysaccharide, Alcian blue was used during fixation. The results obtained show the differences in structural organization of appendages and exosporium in different strains. Canoe-shaped inclusions were revealed in all Br. laterosporus strains, while strain IGM16-92 had a fibrillar capsule as well. Electron microscopy using a dual beam scanning electron microscope Quanta 200 3D provided the information of the spore surface relief without sample treatment (fixation and dehydration). The spores of Br. laterosporus strains had folded surface, unlike the smooth surface of B. cereus and B. thuringiensis spores. The diversity of external spore structures was shown within a species, which may be used for detection of bacteria at the strain level. Optimized procedures for visualization of spore surface by different electron microscopic techniques were discussed.     

Fungal adhesion in aquatic hyphomycetes

The attachment of propagules of aquatic fungi often takes place under turbulent conditions, i.e. spores of marine lignicolous fungi and conidia of freshwater Hyphomycetes. It has been demonstrated that attachment in Lemonniera aquatica is effected by three arms of the conidium making contact with the surface and forming appressoria. In this study the mechanisms of attachment of conidia of 10 aquatic Hyphomycetes to a variety of natural and artificial substrata were followed at the light microscope and scanning electron microscope levels.Five species produced appressoria, while in the remaining species attachment and subsequent retention to the substratum was effected solely by the production of mucilage. Examination of selected appressorium-forming species in the transmission electron microscope showed that conidium attachment involved the production of mucilage in the regions of initial contact and only then were appressoria formed. The ecological significance of appresoiria to the aquatic Hyphomycetes is discussed.     

Scanning electron microscopy of Entomophthora virulenta and Conidiobolus coronatus (Entomophthorales: Entomophthoraceae)

The scanning electron microscope was used to delineate the morphological differences between Entomophthora virulenta and Conidiobolus coronatus isolated from field-infected cabbage and seedcorn maggots, Hylemya brassicae and H. platura. This SEM study supports the removal of C. coronatus from the genus Entomophthora on the basis of microconidial development and resting spores with villose appendages.     

Fossil plants with spores in the sporangia from the Upper Devonian (Frasnian) deposits of northern Timan

Fossil plants from the Upper Devonian (Frasnian) of northern Timan are studied. The sporangia contain well-preserved spores, which were studied in transmitted light using a scanning electron microscope. The genus Gutzeitia S. Snigirevsky, gen. nov. is established. Macroremains and in situ spores of G. timanica (Petros.) S. Snig., comb. nov. and ?Cephalopteris mirabilis (Nath.) Nath. are described. Microspores in Dimeripteris gracilis Schmalh. are identified, and microspores and megaspores in ?Cephalopteris mirabilis are studied for the first time.     

FERRITIN IN THE FUNGUS PHYCOMYCES

The iron-protein ferritin has been purified from mycelium, sporangiophores, and spores of the fungus Phycomyces blakesleeanus. It has a protein-to-iron ratio of 5, a sedimentation coefficient of 55S, a buoyant density in CsCl of 1.82 g/cm³, and the characteristic morphology of ferritin in the electron microscope. Apoferritin prepared from Phycomyces ferritin has a sedimentation coefficient of 18S and consists of subunits of molecular weight 25,000. In the cytoplasm of Phycomyces, ferritin is located on the surface of lipid droplets (0.5–2.0 µ in diameter) where it forms crystalline monolayers which are conspicuous in electron micrographs of sporangiophore thin-sections. Ferritin is found in all developmental stages of Phycomyces but is concentrated in spores. The level of ferritin iron is regulated by the iron level in the growth medium, a 50-fold increase occurring on iron-supplemented medium.     

Osservazioni preliminari al microscopio elettronico a scansione sulle ornamentazioni delle spore di Russula

Abstract

Preliminary observations on spore ornementation of Russula, as seen in the scanning electron microscope. — The spores of 16 species of Russula have been examined in the scanning electron microscope, as a preliminary attempt to see if an accurate examination of the spore surface at the ultrastructural level could reveal details of ornamentation which might be useful for the classification of the many species of this genus. The examination, carried out both on fixed and unfixed specimens, has demonstrated that the spore shape is always round or slightly elliptic, and that the obnormal forms as previously described are probably artifacts. Five main types of ornamentation have been described: single wart-like and single finger-like Protrusion, wart- or finger-like protrusions interconnected by thin ridges, and thick, short « papillae » together with large ridges that run along large tracts of the spore circumference. The type of ornamentation was a constant character in each species.     

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.     

THE FINE STRUCTURE OF STREPTOMYCES VIOLACEORUBER (S. COELICOLOR) : III. The Walls of the Mycelium and Spores

A study of thin sections of hyphae of Streptomyces violaceoruber in the electron microscope showed that the structure of the walls and the mode of formation of cross-walls are similar to those of Gram-positive bacteria. A beaded structure was seen in some regions of the wall, and the significance of this observation is discussed in relation to previous studies of the fine structure of bacterial cell walls. Elements of the intracytoplasmic membrane system appear to be involved in the process of cross-wall formation. The walls of the hyphae of the aerial mycelium divide into two layers before the spores are formed, and only the inner component of the wall grows inwards to form the cross-walls and so delimit the spores. The outer component remains intact for a time and acts as a sheath around the developing spores. Finally the sheath breaks and the spores are liberated. This process is contrasted with the formation of endospores in eubacteria. When the spores germinate, the walls of the germ tubes are continuous with those of the spores.     

Scanning electron microscope observations on the spore of Nosema apis Zander

A simple Epon block fracture technique was used to process spores of Nosema apis for scanning clectron microscope examination. The mature spore was egg-shaped with a pointed anterior pole. The young spore was more elongated. The surface of both stages was smooth. A large number of midgut epithelial cells which harbour N. apis were also observed. The cell surfaces of the epithelial cells were also smooth.     

Insecticidal activity of the crystalline parasporal inclusions and other components of the Bacillus sphaericus 1593 spore complex

Bacillus sphaericus 1593 spore complexes were disrupted by French pressure cell. Fractions recovered from centrifugation of these complexes on 10–50% NaBr gradients were assayed against mosquito larvae and examined using the electron microscope. Crystalline parasporal inclusions were concentrated in the fraction of highest insecticidal activity. The fractions containing sporangium, exosporium, and spores also were insecticidal at a lower level. These results indicate that the crystals are the major source of insecticidal toxin in strains of B sphaericus which produce them.     

LOCALIZATION OF STRUCTURAL POLYMERS IN THE CELL WALL OF NEUROSPORA CRASSA

The distribution and localization of structural polymers in the cell wall of Neurospora crassa has been studied by selective removal and light and electron microscope examination. Observations with the light microscope indicated that each polymer by itself can provide structural integrity to the cell wall. Examination by electron microscopy showed that the cell wall consists of an outer layer of thick fibrils, identified chemically as a glucan-peptide-galactosamine complex, and an inner layer made up of β-1,3 glucan and thin fibrils of chitin.     

Plasmodium: high-voltage electron microscopy

Stereoscopy of thick sections (~1.0 μm) of biological material, Plasmodium gallinaceum and Plasmodium berghei, was investigated with a high-voltage electron microscope using an accelerating voltage of 650 kv. Not only could structural details of the malarial parasites be observed in the thick sections, but stereoscopic examination also revealed the physical relationships of the various subcellular organelles in the parasites. This study indicates that the high-voltage electron microscope is a useful tool for structural analysis of malarial parasites.     

Ultrastructure of laevigate hilate spores in sporangia and spore masses from the Upper Silurian and Lower Devonian of the Welsh Borderland

Spore masses and isolated sporangia, containing laevigate hilate cryptospores attributable to the dispersed taxon Laevolancis divellomedia sensu lato, have been recovered on bulk maceration of Upper Silurian (Pridoli) and Lower Devonian (Lochkovian) deposits from the Welsh Borderland. Detailed morphological, anatomical and ultrastructural analysis, using light microscope, scanning electron microscope and transmission electron microscope techniques, reveals subtle differences between the specimens and they can be grouped into five distinct types. The different groups are distinguished principally by using sporangia-spore mass characteristics, presence or absence of extra-exosporal material and nature of spore-wall ultrastructure. Of the groups, one has a uniformly homogeneous exospore and the other four groups have a bilayered exospore. In the former the spores lack extra-exosporal material and occur in a discoidal sporangium. Of the bilayered groups, two have exospores of homogeneous composition but with the two layers differing in electron density. They occur in discoidal sporangia and spore masses and are distinguished on the presence or absence of extra-exosporal material and differences in the widths of the two layers. Finally, two bilayered groups possess a lamellate inner layer, but vary in presumed sporangial shape. Elongate sporangia have spores with concentric continuous lamellae, lacking further ultrastructure. In contrast, spores from a discoidal spore mass have white-line-centred, presumably tripartite, lamellae which are laterally discontinuous, overlapping and irregularly spaced. These findings, which suggest that morphologically similar spores were produced by a number of plant taxa, have important implications regarding the assessment of early land-plant diversity. The affinities of hilate cryptospore-producing plants are unknown and problematic, particularly as no extant non-angiosperm plants produce dyads, other than through meiotic irregularity, and spore-sporangial characters have no exact counterpart in coeval plants. Studies of specimens with in situ hilate cryptospores suggest that they derive from rhyniophytoids, i.e. plants that resemble the simplest of vascular plants but lack evidence of vascular tissue, although hilate cryptospore-containing examples show no axial branching. It might be argued, based on evidence from spore wall ultrastructure, that some of the plants have more in common with lycopsids and filicopsids than bryophytes, a surprising finding bearing in mind the stratigraphic distribution of hilate cryptospores-dyads and inferences that the producers were bryophyte-like. Detailed studies of wall structure in the hilate cryptospores permit consideration of spore wall development. It is suggested that extra-exosporal material derives from a tapetum and is thus produced by the diploid sporophyte. The white-line-centred lamellae in a single specimen provide the earliest evidence for the presence of such structures in early land plant spores and provide further evidence that sporopollenin deposition on such structures is the most primitive mode of sporopollenin deposition among land plants.     

Lower Devonian coalified sporangia from Shropshire: Salopella Edwards & Richardson and Tortilicaulis Edwards

A diverse assemblage of coalified sporangia from Lochkovian/Gedinnian, fluvial rocks imicrornatus-newportensis Spore Biozone) contains fusiform forms assignable to Salopella Edwards & Richardson and Tortilicaulis Edwards. Both show bifurcation within some sporangia. In specimens assigned to Salopella cf. marcensis , longitudinal dehiscence produces two equal valves revealing spores resembling Aneurospora , although trilete marks may be difficult to distinguish. The spores are in two forms with distal ornament of either coarse or fine coni, each occurring in separate sporangia. A further sporangium of Salopella shape contains distally apiculate, hilate cryptospores. Elongate sporangia exhibiting spiralling of superficial cells and sometimes gross twisting, are placed in a new species of Tortilicaulii, T. offaeus. Anatomical data include details of irregularly thickened epidermal cells, sporangial wall and in situ spores. The latter are trilete, equatorially thickened and highly distinctive, because the entire exospore surface is covered with grana, a feature not common in dispersed spores of this age. The nomenclatural and curatorial problems associated with these small, coalified fossils, whose anatomical examination by scanning electron microscope necessitates elimination of gross morphology, are discussed.     

孢粉药材及花类药材的孢粉形态(续)

 利用光学显微镜和扫描电子显微镜对10种孢粉药材及花类药材进行了孢子或花粉的显微和亚显微研究,为孢粉药材及花粉药材的鉴别提供了新的方法和科学依据。