Electron Microscopic Examination of Corynebacterium ovis

Corynebacterium ovis (C. pseudotuberculosis) was examined by electron microscopy after being subjected to various methods of fixation. The organism exhibited a fine structure similar to other corynebacterial species in the appearance of its cell wall, plasma membrane, nuclear apparatus, cytoplasmic matrix, wealth and complexity of intracytoplasmic membrane systems, and polyphosphate granules. An outstanding structural feature was the existence of an electron-dense, floccular layer external to the cell wall which both ligroin and acetone-methanol extractions demonstrated to be the previously postulated surface lipid of this organism. The only variations in structure evident between virulent and attenuated strains was a quantitative difference in the thickness and appearance of the surface lipid. The observation of this layer provided a basis for explaining the surface properties of C. ovis, with particular respect to its clumping capacity in suspension, the waxiness of its growth on solid media, and its ability to grow as a pellicle on suitable liquid media. The variation in the visible amount of surface lipid between the virulent and avirulent strains adequately explained the divergence of these three surface properties between the strains.     

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.     

Preparation of Whole Rabbit Knee Joints for Microscopic Examination

A paraffin embedding method to prepare whole rabbit knee joints for histological examination is described. This method provides good quality microscopic sections thin enough for the study of cellular detail and does not require prolonged processing. When examining pathologic changes in experimental arthritis, it is advantageous to be able to examine the intact joint with the structural relations of the joint components preserved. Sections of the whole joint provide numerous areas where bone, cartilage and synovium are contiguous for examination. Having obtained poor results using methods recommended for small bony specimens, we modified several existing procedures to obtain a reliable method for preparing excellent microscopic sections of the whole rabbit knee joint.     

镜检脑脊液白细胞定量计数方法改进的研究

目的:建立手工法镜检脑脊液白细胞定量的快速、准确、直观的检测方法,并验证该方法比传统方法准确并可靠性高。方法:无菌条件下采集患者脑脊液标本230例,分别采用中华人民共和国卫生部医政司出版的《全国临床检验操作规程》上的规程法、定量法。两种方法对于同一样本进行白细胞数计数,实验重复3次,用单因素方差分析进行统计学比较。结果:镜检脑脊液白细胞计数定量的方法比传统的方法(P>0.05,sd,cv值更小)更准确精密可靠。结论:计数定量的方法比规程法更准确,精密,可靠。     

Immuno-Electron Microscopy of the Morphogenesis of Mumps Virus

The fine structure of mumps virus-infected chick embryo fibroblastic cells was examined sequentially after viral inoculation. Intracytoplasmic nucleoprotein strands, similar to those described for parainfluenza viruses, were detectable in small aggregates between 36 and 48 hr. The peripheral strands of this viral component lie beneath and along an antigenically altered bulging portion of the cell membrane. The outermost strands are consistently parallel to the differentiated segment of the plasma membrane, which is invariably associated with surface projections. As has been found with other myxoviruses, mumps virus replicates by budding from the cell surface. The virus particle, roughly spherical in shape, has a size ranging from 1,000 to 8,000 A. Filamentous forms are rarely observed in the present culture system. Ferritin-conjugated antibody specifically labels the cytoplasmic nucleoprotein, the modified cell membrane, and the virus particle. Intranuclear inclusions of low electron density and morphologically different from those described in measles virus-infected HeLa and amnion cells were observed in the nucleus of several infected cells. Immuno-electron microscopic observations suggest that the nucleoprotein synthesis rate exceeds that of cell membrane differentiation into viral envelope. This difference results in the accumulation of viral nucleoprotein in large intracytoplasmic masses which can be demonstrated by electron microscopy.     

Microscopic and Calorimetric Assessment of Freezing Processes in Uterine Fibroid Tumor Tissue

The use of cryosurgery in the treatment of uterine fibroids is emerging as a possible treatment modality. The two known mechanisms of direct cell injury during the tissue freezing process are linked to intracellular ice formation and cellular dehydration. These processes have not been quantified within uterine fibroid tumor tissue. This study reports the use of a combination of freeze-substitution microscopy and differential scanning calorimetry (DSC) to quantify freeze-induced dehydration within uterine fibroid tumor tissue. Stereological analysis of histological tumor sections was used to obtain the initial cellular volume (V(o)) or the Krogh model dimensions (deltaX, the distance between the microvascular channels = 15.5 microm, r(vo), the initial radius of the extracellular space = 4.8 micro m, and L, the axial length of the Krogh cylinder = 19.1 microm), the interstitial volume ( approximately 23%), and the vascular volume ( approximately 7%) of the fibroid tumor tissue. A Boyle-van't Hoff plot was then constructed by examining freeze-substituted micrographs of "equilibrium"-cooled tissue slices to obtain the osmotically inactive cell volume, V(b) = 0.47V(o). The high interstitial volume precludes the use of freeze-substitution microscopy data to quantify freeze-induced dehydration. Therefore, a DSC technique, which does not suffer from this artifact, was used to obtain the water transport data. A model of water transport was fit to the calorimetric data at 5 and 20 degrees C/min to obtain the "combined best fit" membrane permeability parameters of the embedded fibroid tumor cells, assuming either a Krogh cylinder geometry, L(pg) = 0.92 x 10(-13) m(3)/Ns (0.55 microm/min atm) and E(Lp) = 129.3 kJ/mol (30.9 kcal/mol), or a spherical cell geometry (cell diameter = 18.3 microm), L(pg) = 0.45 x 10(-13) m(3)/Ns (0.27 microm/min atm) and E(Lp) = 110.5 kJ/mol (26.4 kcal/mol). In addition, numerical simulations were performed to generate conservative estimates, in the absence of ice nucleation between -5 and -30 degrees C, of intracellular ice volume in the tumor tissue at various cooling rates typical of those experienced during cryosurgery (< or =100 degrees C/min). With this assumption, the Krogh model simulations showed that the fibroid tumor tissue cells cooled at rates < or = 50 degrees C/min are essentially dehydrated; however, at rates >50 degrees C/min the amount of water trapped within the tissue cells increases rapidly with increasing cooling rate, suggesting the formation of intracellular ice.     

应用原子力显微镜对玉米染色质纤丝及染色体骨架的研究(英文)

应用原子力显微镜对经化学方法预处理的玉米染色体超微结构进行了研究。原子力显微镜观察的结果揭示,经30%醋酸处理的玉米染色体表面呈现出不均一的颗粒状结构。当缩小扫描范围后,在染色体表面发现直径分别为30和100 nm的两种染色质纤丝。100 nm的染色质纤丝由30 nm染色质纤丝螺旋缠绕而成。在经25%胰酶处理的玉米染色体上发现了两种类似的螺旋状染色质纤丝结构,其直径分别为30和100～150 nm。较细的染色质纤丝螺旋缠绕成较粗的纤丝,进而构成整个染色体。经低离子浓度溶液抽提,用原子力显微镜观察到了玉米染色体的染色体骨架结构。这种染色体骨架呈不规则的纤维网状,这些网状纤维在染色体中部显得较为紧密,在染色体的边缘则显得较松散。这一结果暗示染色体是由不同级别的染色质纤丝螺旋缠绕构成,为染色体的多级螺旋结构假说提供了新的证据。同时发现染色体骨架并不是呈轴样的结构存在,而是保留了染色体的基本形态,这种骨架形状也许是由分散在染色体中的染色体骨架蛋白在低离子浓度溶液抽提的过程中凝缩形成的。     

镜检法在腹腔内接种包虫病动物模型中的应用

目的建立一种简单快速观察腹腔内接种包虫病动物模型的方法。方法按2000个原头节／mL剂量,用多房棘球蚴对灰仓鼠进行腹腔接种,采用肉眼观察法和显微镜镜检法在第10天,15天,18天,22天,39天和60天观察灰仓鼠腹腔内包囊、包囊液和原头节的生长情况;设空白对照组10只。结果通过肉眼观察和显微镜镜检,发现灰仓鼠在接种后的第18天有原头蚴生长;第15天,18天和22天包囊增大、增多;第39天有成熟原头节胚基生长;第60天有不同发育程度的原头蚴。随着接种时间的延长,包囊重量与传代时间成正比。结论本试验为制备包虫病动物模型提供了简单快速的观察方法。     

Assessing the Application of Tissue Microarray Technology to Kidney Research

Tissue microarray (TMA) is a new high-throughput method that enables simultaneous analysis of the profiles of protein expression in multiple tissue samples. TMA technology has not previously been adapted for physiological and pathophysiological studies of rodent kidneys. We have evaluated the validity and reliability of using TMA to assess protein expression in mouse and rat kidneys. A representative TMA block that we have produced included: (1) mouse and rat kidney cortex, outer medulla, and inner medulla fixed with different fixatives; (2) rat kidneys at different stages of development fixed with different fixatives; (3) mouse and rat kidneys with different physiological or pathophysiological treatments; and (4) built-in controls. As examples of the utility, immunostaining for cyclooxygenase-2, renin, Tamm Horsfall protein, aquaporin-2, connective tissue growth factor, and synaptopodin was carried out with kidney TMA slides. Quantitative analysis of cyclooxygense-2 expression in kidneys confirms that individual cores provide meaningful representations comparable to whole-kidney sections. These studies show that kidney TMA technique is a promising and useful tool for investigating the expression profiles of proteins of interest in rodent kidneys under different physiological and pathophysiological conditions. (J Histochem Cytochem 58:413–420, 2010)     

Quantitative Examination of Tissue Concentration Profiles Associated with Microdialysis

Spatial solute concentration profiles resulting from in vivo microdialysis were measured in rat caudate-putamen by quantitative autoradiography. Radiolabeled sucrose was included in the dialysate, and the tissue concentration profile measured after infusions of 14 min and 61.5 min in an acute preparation. In addition, the changes in sucrose extraction fraction over time were followed in vivo and in a simple in vitro system consisting of 0.5% agarose. These experimental results were then compared with mathematical simulations of microdialysis in vitro and in vivo. Simulations of in vitro microdialysis agreed well with experimental results. In vivo, the autoradiograms of the tissue concentration profiles showed clear evidence of substantial differences between 14 and 61.5 min, even though the change in extraction fraction was relatively small over that period. Comparison with simulated results showed that the model substantially underpredicted the observed extraction fraction and overall amount of sucrose in the tissue. A sensitivity analysis of the various model parameters suggested a tissue extracellular volume fraction of approximately 40% following probe implantation. We conclude that the injury from probe insertion initially causes disruption of the blood-brain barrier in the vicinity of the probe, and this disruption leads to an influx of water and plasma constituents, causing a vasogenic edema.     

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.     

New Technique for Microscopic Examination of the Fouling Community of Submerged Opaque Surfaces

The fouling film formed on surfaces of opaque materials submerged in seawater could be almost quantitatively removed for microscopy by application of a Parlodion film.     

A Vital Microscopic Description of the Effects of Electrical Stimulation of Bone Tissue

An implantable, titanium, optical chamber that allows vital microscopic observations of bone tissue during continuous electrical stimulation is described. Tissue reactions to direct current stimulation of a defined bone tissue compartment may be repeatedly observed and recorded on film for indefinite follow-up periods. In this study 5, 20 or 50 μA direct current were applied to chambers inserted into the rabbit tibia. The bone tissue was found to increase in volume after stimulation with 5 and 20 μA, while 50 μA caused bone resorption. Fat-cell concentration was independent of the current levels as used in the present study. Vital microscopy revealed no signs of acute microvascular alterations in the hours following onset of the current. Over an eleven-week follow-up, vessels close to bone borders were found to increase in size from 15–25 microns to 30–50 microns, independent of the level of stimulation. The amount of vessels increased after stimulation with 5 and 20 μA, while in the 50 μA group the vessels decreased to 50% or less of the vascular density observed before start of the stimulation.     

Interaction between Venturia inaequalis and Apple Callus Tissue Cultures: an Electron Microscopic Study

Untrastructural interactions between Venturia inaequalis and callus cultures from scab susceptible and resistant apple varieties, were similar. Host cell wall changes, appositions, and invagination of host plasmamembrane at sites of close contact with fungal hyphae were regularly observed. The ultrastructural observations are described and discussed. The host cell alterations as well as many fungal structures corresponded to those known in young leaves of susceptible apple varieties.     

Electron Microscopic Labeling of Intercellular Spaces in Isolated Tissue Specimens with Horseradish Peroxidase

For electron microscopic study of cellular interactions it can be useful to mark the intercellular space with an electron opaque substance. By doing so, intercellular contacts, surface membrane fusions and membrane defects, as well as junctional associations, can be demonstrated much better (Gilula 1974). Ruthenium red (Luft 1971) or lanthanum (Revel and Karnovsky 1967) have often been used for this purpose. Both substances can be applied by perfusion or by diffusion technics. Unfortunately both show a rather irregular and inconstant distribution in the interstices of tissues, regardless of which technic has been applied (Luft 1971, Gebbers and Otto 1974).     

Simultaneous Histological Preparation of Bone, Soft Tissue and Implanted Biomaterials for Light Microscopic Observations

Block specimens of formalin fixed bone, soft tissue and endosseous implanted biomaterials can be successfully embedded in polymethyl methacrylate by employing vacuum desiccation during the dehydration steps and refrigeration during the infiltration step. One-hundred-micrometer histological sections can be obtained from the cured polymethyl methacrylate blocks by cutting with a low concentration diamond wafering blade on a Buehler Isomet Circular Low Speed Saw using Buehler Isocut fluid. The sections can be readily stained and details of individual cells studied by light microscopy, thus allowing interpretation of the relationship between biomaterial and surrounding tissues. The advantage of this method is that it allows observation of the entire specimen in situ. The details of the procedure are presented.     

Repair of Injury in Freeze-Dried Salmonella anatum

Repair of injury induced by freeze-drying Salmonella anatum in nonfat milk solids occurred rapidly after rehydration. Injury in surviving cells was defined as the inability to form colonies on a plating medium containing deoxycholate. Death was defined as inability to form colonies in the same medium without this selective agent. The rate of repair of injury was reduced by lowering the temperature from 35 C to 10 C and was extremely low at 1 C. Repair was independent of influence of pH between 6.0 and 7.0. Repair did not require synthesis of protein, ribonucleic acid, or cell wall mucopeptide, but did require energy in the form of adenosine triphosphate (ATP) synthesized through oxidative phosphorylation. The requirement for ATP was based on dinitrophenol or cyanide interference with repair. Dinitrophenol activity was pH-dependent; no repair occurred at pH 6.0 and some repair was observed at pH 6.5 and above. Injured cells were extremely sensitive to low concentrations of ethylenedinitrilotetraacetate. This indicated that freeze-drying injury of S. anatum may involve the lipopolysaccharide portion of the cell wall and that repair of this damage requires ATP synthesis.     

Interleukin-19 Mediates Tissue Damage in Murine Ischemic Acute Kidney Injury

Inflammation and renal tubular injury are major features of acute kidney injury (AKI). Many cytokines and chemokines are released from injured tubular cells and acts as proinflammatory mediators. However, the role of IL-19 in the pathogenesis of AKI is not defined yet. In bilateral renal ischemia/reperfusion injury (IRI)-induced and HgCl₂-induced AKI animal models, real-time quantitative (RTQ)-PCR showed that the kidneys, livers, and lungs of AKI mice expressed significantly higher IL-19 and its receptors than did sham control mice. Immunohistochemical staining showed that IL-19 and its receptors were strongly stained in the kidney, liver, and lung tissue of AKI mice. In vitro, IL-19 upregulated MCP-1, TGF-β1, and IL-19, and induced mitochondria-dependent apoptosis in murine renal tubular epithelial M-1 cells. IL-19 upregulated TNF-α and IL-10 in cultured HepG2 cells, and it increased IL-1β and TNF-α expression in cultured A549 cells. In vivo, after renal IRI or a nephrotoxic dose of HgCl₂ treatment, IL-20R1-deficient mice (the deficiency blocks IL-19 signaling) showed lower levels of blood urea nitrogen (BUN) in serum and less tubular damage than did wild-type mice. Therefore, we conclude that IL-19 mediates kidney, liver, and lung tissue damage in murine AKI and that blocking IL-19 signaling may provide a potent therapeutic strategy for treating AKI.