Purification and partial characterization of the exotoxin of Corynebacterium ovis.

1. The toxin from Corynebacterium ovis, a phospholipase D (sphingomyelin phosphodiesterase D) that acts on 2-lysophosphatidylcholine and sphingomyelins, was purified by about 400-fold to homogeneity as judged by several criteria. [The EC number of the toxin (EC 3.1.4.41) has been allotted by the Nomenclature Committee of IUB, but has not yet been published.] 2. A new assay method performed in vitro, based on inhibition by the toxin of erythrocyte lysis by staphylococcal beta-haemolysin, was developed to facilitate the purification. 3. The toxin was found to be a basic (pI9.1) glycoprotein of mol.wt. 14,500 +/- 1,000. 4. The amino acid composition of the toxin was highly reminiscent of that of collagen, since it contained hydroxyproline, hydroxylysine and a high proportion of glycine, but preliminary tests showed no other similarities to collagen or proteins with similar compositions.     

Rapid Three-Dimensional Reconstruction at the Light Microscopic Level and a Technique for Re-Embedding the Same Semithin Sections for Electron Microscopic Examination

Rapid three-dimensional reconstruction of serial sections at the light microscopic and ultrastructural levels was accomplished using a two-step technique. Fixed specimens were embedded in Epon and 1 μm sections were cut and placed on glass slides. One of every four sections was drawn onto transparency film for rapid three-dimensional reconstruction. The semi-thin sections were re-embedded in Epon and sectioned at 90 nm for examination in the electron microscopy.     

Electron Microscopic Examination and Quantitation of Rhapidosomes,Viruses and Bacteria Concentrated at an Aqueous-Air Interface

Small volumes of suboptimal particle concentrations for electron microscopy may easily provide at least the required 3.4 × 10⁷ particles per cubic centimeter for this purpose when concentrated 10 to 1000-fold at an aqueous-air interface. As a result, ascending droplets of microorganisms derived from bursting bubbles were adsorbed to an Inverted agar surface strategically positioned above the reaction vessel. The agar surface was subsequently pseudoreplicated in a solution of uranyl-magnesium acetate for electron microscopic examination and quantitation.     

Electron Microscopic Observations of Rabbit Antibodies

Electron micrographs were obtained showing the individual, shadow-cast macromolecules from solutions of purified anti-p-azobenzoate rabbit antibody and of normal γ-globulin. The two materials look alike and consist mainly of asymmetrical rod-like particles about 30 to 40 A in diameter. Lengths are not constant but the weight average is about 250 A for the antibodies and about 200 A for the γ-globulin. The average observed dimensions are reasonably consistent with values deduced from physical-chemical methods, although the shape is more nearly that of a cylindrical rod rather than the ellipsoid employed in hydrodynamical theory. Mixtures of antibody and specific dihaptenic dye were examined in attempts to establish the mode of the specific aggregation. At the high dilutions necessary for electron microscopy (0.1 mg./ml.), the effect of the dye was small and tended to be masked by non-specific aggregation on drying. The evidence suggests that under these conditions the specific reaction involves an end-to-end aggregation of the elementary particles to produce a weight average length about twice that of the pure antibody.     

浮游病毒的电镜观察

直接用戊二醛固定水样中的浮游病毒,通过超速离心使已固定的浮游病毒沉淀到覆有Formvar膜和碳支持膜的铜网上,经醋酸双氧铀染色后,利用透射电镜对湖水中的浮游病毒进行观察．结果可观察到球形、杆状和蝌蚪状等形态各异的浮游病毒颗粒及球形病毒的囊膜子粒、杆状病毒的核衣壳、具有不同尾部的蝌蚪状病毒等的精细超微结构．从而建立了一种简便、快捷和高效研究浮游病毒的电镜方法．     

Effects of Lysozyme and Inorganic Anions on the Morphology of Streptococcus mutans BHT: Electron Microscopic Examination

The effects of hen egg white lysozyme and the inorganic salt sodium thiocyanate on the integrity of Streptococcus mutans BHT were studied by transmission electron microscopy. Both control cells and cells exposed to NaSCN possessed thick outer cell walls and densely staining inner cell walls juxtaposed to the plasma membranes. In the presence of NaSCN, however, the S. mutans BHT nucleoid was coagulated into thick electron-dense filaments. Exposure of S. mutans BHT to 150 μg of hen egg white lysozyme per ml resulted in the progressive destruction of both the cell walls and the plasma membranes. The enzyme appeared to affect the region of the cell wall septum, and exposure to 150 μg of hen egg white lysozyme per ml for as short a time as 10 min resulted in visible morphological cell wall alterations. At 30 min, ultrastructural observations revealed that the majority of the cells were in the process of expelling a portion of their cytoplasmic contents from the septal and other regions of the cells at the time of fixation. After 3 h of incubation in the presence of this high lysozyme concentration, gelled protoplasmic masses, which were free from the cells, were evident. In addition, extensive damage to the outer and inner cell walls and to the plasma membranes was apparent, although the cells maintained their shape. On some areas of the cell surface, the outer cell wall and plasma membrane were completely absent, whereas at other locations the outer cell wall was either split away from the inner cell wall and plasma membrane or distended from an area free of inner cell wall and plasma membrane. Upon addition of NaSCN to the hen egg white lysozyme-treated cells, both the gelled protoplasmic masses and the damaged cells exhibited an exploded appearance and existed as membrane ghosts, cell wall fragments, or dense aggregates of cytoplasmic components. The effects of a low lysozyme concentration (22.5 μg/ml) on S. mutans morphology were less pronounced at short incubation times (i.e., 10 and 30 min) than those that were observed with a high enzyme concentration; however, breaks in the cell walls and dissolution of the plasma membranes with resulting cell lysis were visible after a prolonged (3-h) incubation and after subsequent addition of NaSCN.     

An Electron Microscopic Study of Erythrophagocytosis

The present study describes a submicroscopic surface fragmentation of erythrocytes which occurs in the ascitic fluid of rats bearing the Novikoff ascites hepatoma. The resulting fragments attach to the surface of macrophages and are phagocytized by pseudopod formation. Plasma membrane in the region of these phagocytosis vacuoles appears to condense into electron-opaque material, suggesting an alteration in its physicochemical state. Stages in intracellular digestion of intact erythrocytes or small fragments within the phagocytosis vacuoles are illustrated; no particles resembling ferritin are observed. The phagocytosis vacuoles possess high levels of acid phosphatase activity. They may be called phagosomes, a type of lysosome. There is no indication of a connection between phagosomes and other formed cytoplasmic organelles. Small vacuoles of the order of 80 mµ in diameter, which may represent pinocytosis vacuoles, are present in the cytoplasm and some appear to be in contact with the phagosome membrane, reminiscent of observations of Rose with HeLa cells.     

Some Observations on Surface Lipids of Virulent and Attenuated Strains of Corynebacterium ovis

S ummary . Observations on the growth of virulent and attenuated strains of Corynebacterium ovis on solid and liquid media, together with quantitative differences in the amount of easily extractable lipid from two of these strains, support the hypothesis that surface lipids are concerned with pathogenicity and virulence of C. ovis. Attenuation was not accompanied by loss of a toxic principle contained in the crude lipid.     

DNA homology study of Corynebacterium diphtheriae v. gravis groups I, II and III, Corynebacterium ulcerans and Corynebacterium pseudotuberculosis (ovis)

The homology of genomes within Krylova 's groups I, II and III of C. diphtheriae, including toxigenic C. diphtheriae and their nontoxigenic precursors within the same group, was confirmed by the method of DNA/DNA molecular hybridization; the homology of DNA within the groups was 89-103%, the thermostability of heteroduplexes being high (on the level of homoduplexes ). The heterogeneity of genomes within these 3 groups of cultivar gravis was confirmed, which made it possible to consider C. diphtheriae, groups I, II and III, to belong to different, though closely related species; in intergroup hybridization the homology of DNA varied, as a rule, between 66% and 73%, while the thermostability of heteroduplexes was low: delta T50 was -3 degrees C to -6 degrees C. The differences in genomes (on the level of different species) between 3 groups of C. diptheriae v. gravis on one hand and C. diphtheriae v. mitis C7 (-) tox- and its convertant C7 (beta) tox+ of phage tox+ on the other hand (DNA homology being 56-62%), as well as between C. diphtheriae v. intermedius No. 328 tox+ on one hand and the representatives of 3 groups of C. diphtheriae v. gravis and C. diphtheriae v. mitis, strain C7 (beta) tox+, on the other hand (DNA homology being 42-43%) were revealed. The heterogeneity of genomes (on the level of different genera) was revealed between C. diphtheriae strains, cultivars gravis (groups I, II and III), mitis (C7(-) tox- and C7 (beta) tox+) and intermedius (No. 328 tox+) on one hand and C. ulcerans and C. pseudotuberculosis (ovis) strains on the other hand; DNA homology was 11-17% for C. ulcerans and 22-26% for C. pseudotuberculosis (ovis), the thermostability of heteroduplexes being at the lowest level (delta T50 was -11 degrees C to -13 degrees C). As a result, C. diphtheriae, classified by Bergey as a single species, was found to comprise 5 species detected by means of marking in accordance with their phenotypical features and genome structure, carried out by the method of DNA/DNA molecular hybridization; among these species were group I, II and III strains of cultivar gravis, strain C7 of cultivar mitis and strain No. 328 of cultivar intermedius. C. ulcerans and C. pseudotuberculosis (ovis) strains investigated in this study can possibly be placed outside the genus including 5 C. diphtheriae species.     

粘质沙雷氏菌的电镜观察

用扫描电镜和透射电镜对粘质沙雷氏菌(Serratia marcescens)的临界点干燥标本和负染标本作了观察,均可见到细胞表面常有一至二个直径为0.12～0.24μm的颗粒。在超薄切片中颗粒则有两种不同的结构:一种是外膜泡(Outer membrane vesicle);另一种是致密体(Dense body)。致密体可能是一种分泌性颗粒,它既不是内部的贮存物,也不似外部进来的异物。它们看来形成于细胞质内后分泌到细胞外。在细菌中这是一种罕见现象。有关致密体的化学性质和功能尚不清楚。文中指出粘质沙雷氏菌的纲胞表面也具有茂密的菌毛(Pili)。     

Electron Microscopic Study of a Slime Layer

Slime layers are being studied in our laboratories in an attempt to understand their functions in the control of pollution in natural streams. A method for fixing, staining, and embedding microorganisms in the intact slime has been developed. In this method, epoxy resin discs are placed in a holder and are introduced into a simulated stream. After various periods of time the discs are punched out of the holder into the fixative. The disc with the attached slime is fixed, stained (4% osmium tetroxide plus ruthenium red), dehydrated, and embedded in epoxy resin so that thin sections can be cut through the vertical plane of the slime mass. Such thin sections permit detailed examination of the attached layer, the surface-slime interface, the spatial relationships between cells in the vertical slime structure, and the strands of extracellular material between and around cells. No special attachment structures were noted as the cells appeared to be attached to the surface by extracellular material alone. This material was observed in strands and netlike forms between cells which are positioned 1 to 4 mum apart in the slime.     

Microscopic Examination of Stools After Partial Gastrectomy

The present study was undertaken to determine the frequency with which undigested meat fibres in the stool could be found after partial gastrectomy. A sample of stool was examined in 61 patients who had undergone a partial gastrectomy and in 92 control patients. The age ranges and average ages of both groups were similar. The stools of 77 of the 92 control patients contained well-digested meat fibres, 14 showed partially digested fibres, and one patient had completely undigested meat fibres. In the postgastrectomy group, 44 of 61 patients had well-digested meat fibres and 17 showed partially digested fibres in their stools. None showed undigested fibres. The present study demonstrated that the finding of undigested meat fibres is as infrequent in postgastrectomy patients as in control patients. If undigested meat fibres are found in the stools of postgastrectomy patients, it is suggestive of pancreatic exocrine insufficiency.     

Scanning Electron Microscopic Studies of Candida albicans

A scanning electron microscopic study of selected morphological stages of Candida albicans is presented. Stages represented are budding yeast cells, mycelial-like forms, chlamydospores, germ tube formation, and an unusual rough cell type.