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In this work, nanometer HA crystals have been synthesized via wet chemical precipitation and characterized. This research studies how key synthesis parameters affect the size and phase purity of the produced HA. Characterization work was carried out using X-ray powder diffraction method and scanning electron microscopy for phase identification and particle sizing, respectively.
Phase-dependent forms of microscopy (such as phase contrast, interference, or polarization microscopy) have been used for many years; the amount of phase retardation produces images based mainly on index of refraction. We have developed a microscope that forms images that depend on small differences in extinction for different forms of incident polarized light. By modulating the polarization of light incident on the sample and digitally recording the difference in intensities of transmitted light, we obtain images which specifically reveal either ordered linear structures or chiral (right- or left-handed) structures. Linearly polarized light, incident alternately with two perpendicular directions of polarization, forms images of structures which have linear order or linear orientation. Right-handed and left-handed circularly polarized light incident alternately on a sample forms images of chiral structures. Structures with neither linear order nor chirality are essentially invisible. Thus, images based on linear dichroism, circular dichroism, and linear and circular differential scattering can be used to detect specific types of structures which may be difficult to observe by conventional methods. We have used such 'linear and circular differential imaging' to study the structure of the nucleolus (the site of RNA synthesis) in live primary spermatocytes of Drosophila when they are transcriptionally active or inactive. Some inactive nucleoli are bipartite, with two distinct structures visible by differential scattering of both linearly and circularly polarized light. The active nucleolus is a single domain; it is clearly distinguished from part of the Y chromosome, and it shows different internal structure with linearly and circularly polarized light. Thus, polarization-dependent images reveal structures which can be associated with the transcriptional activity of cells.
Scanning electron microscopy of isolated epidermal cells from newborn mice grown in vitro showed that the cultures consisted of several morphologically different types of cells. Young cultures had many smooth, round cells while older cultures contained more cells with rough or ruffled surface and a varying number of flat, irregular cells. Probably, the various cell types corresponded to different stages of differentation (keratinization). Cultures that were grown in the presence of retinyl acetate (12.5 microgram/ml) had more round and smooth cells after several days in vitro than the controls. This could indicate that retinyl acetate delayed or altered cell differentiation. Scanning electron microscopy of the cultures consistently showed that the cells were situated at different layers. The apparent monolayer seen by phase contrast microscopy therefore seems to be an optical phenomenon due to projection of the cells onto the same plane.
The category of provider-performed microscopy was defined in the Federal Register to provide a unique regulatory approach for bright-field and phase-contrast microscopy performed by a physician, dentist, or midlevel practitioner examining labile specimens. A CLIA certificate for this category of testing is available, which also permits the performance of waived tests. Because these provider-performed microscopy procedures do not have quality-control materials available, special challenges are encountered in establishing and monitoring such testing within a hospital. Vigilance is required to maintain a provider-performed microscopy program within a hospital.
Five strains of acetogenic bacteria were isolated by selective enrichment from the rumen of a mature Hereford crossbred steer fed a typical high forage diet. Suspensions of rumen bacteria, prepared from contents collected 7 h postfeeding, blended and strained through cheesecloth, were incubated in a minimal medium containing 10% clarified rumen fluid under either H2:CO2 (80:20) or N2:CO2 (80:20) headspace atmosphere. The selection criterion was an increment of acetate in the enrichments incubated under H2:CO2. Periodically, the enrichment broths were plated onto agar media and presumed acetogenic bacteria subsequently were screened for acetate production. Selected acetogenic bacteria utilized a pressurized atmosphere of H2:CO2 to form acetate in quantities 2 to 8-fold higher than when grown under N2:CO2. All presumptive acetogenic isolates were derived from either the 10(-7) or 10(-8) dilutions of rumen contents. All 5 strains were Gram-positive rods, and all utilized formate, glucose and CO. One strain required, and all were stimulated by, rumen fluid. No spores were observed with phase-contrast microscopy and two strains were motile. No methane was detected in the headspace of pure cultures grown under either gas phase. The isolation of these bacteria indicates that acetogenic bacteria are inhabitants of the rumen of the bovine fed a typical diet and suggests that they may be participants in the utilization of hydrogen in the rumen ecosystem. Strain 139B (= ATCC 43876) is named Acetitomaculum ruminis gen. nov., sp. nov. and is the type strain of this new species.
Rapid detection of CMV-DNA in urine specimens by dot-blot hybridization was compared to conventional virus isolation and to virus identification using solid-phase immunoelectron microscopy (SPIEM). To detect viral DNA, 32P-labeled EcoR1 J fragment of CMV-DNA was used as a probe in the hybridization assay. In addition, DNA extracted from infected human embryo fibroblasts (amplified DNA) was also hybridized to the same probe. Urine specimens were obtained from 10 renal transplanted patients, seven premature infants, three family members, and five children suspected of CMV infection. CMV was isolated from 10 urine specimens and SPIEM detected viral particles in nine specimens. Ten positive samples were identified as such by hybridization with DNA extracted directly from urine specimens, while hybridization with amplified DNA yielded 17 positives. Only in one urine specimen, positive by virus isolation and SPIEM, DNA was not detected by the hybridization assays. Elevated IgG or IgM-specific antibodies were found in 10 patients. Hybridization with amplified DNA proved the most sensitive and relatively rapid assay, as compared with direct DNA detection in urine, tissue culture isolation, SPIEM, or serologic tests.
We demonstrate experimentally that correct phase imaging without 2pi ambiguity is obtainable in digital holography by using a multiwavelength approach in the microscope configuration. We describe a general approach for removing chromatic aberrations and for controlling the pixel size of the reconstructed phase image in multiwavelength digital holography when the Fourier transform method is adopted for the numerical reconstruction of digital holograms. The retrieved phase is affected by the unavoidable, unwanted chromatic aberration. The correct phase can be obtained by evaluating the phase from the reference holograms reconstructed at different wavelengths to compensate for the chromatic aberration.
Mitochondrial movements in Saccharomyces cerevisiae (Sc) zygotes were monitored with phase-contrast microscopy and compared to known mitochondrial inheritance systems. The mitochondria of Sc were convincingly identified by integrated use of phase-contrast, cytochemical and electron microscopic observations. Mitochondria in Sc appear to move by saltatory jumps, which appear to be oriented towards movement of mitochondria into developing buds. Tracking of mitochondria of different genotypes was made possible by positive identification of each mitochondrial population before zygosis, and by the low degree of mixing (less than 10%) of mitochondrial populations before first bud septation. A grande by grande cross demonstrated equal numbers of mitochondria from each haploid moving into the first zygotic bud. A grande by neutral petite cross gave a 2:1 ratio of grande to petite mitochondria. However, a grande by suppressive petite cross gave equal numbers of grande and petite mitochondria. Using drug resistance systems, a comparison was made of highly biased (97%) and moderately biased (71%) chloramphenicol resistant inheritance patterns. In both cases, the ratios of drug resistant to sensitive mitochondria were 1:1. When numbers of mitochondria moving into an individual bud were compared to the phenotypic content of the clone of that bud, no model could be constructed which could predict the latter from the former. The data indicate (with the exception of the neutral petite by grande cross) that the numbers of each mitochondrial type "inserted" into the first zygotic bud are equal, regardless of the degree of asymmetry of inheritance of mitochondrial markers.
Liquid-ordered phase (lo phase) of lipid membranes has properties that are intermediate between those of liquid-crystalline phase and those of gel phase and has attracted much attention in both biological and biophysical aspects. Rafts in the lo phase in biomembranes play important roles in cell function of mammalian cells such as signal transduction. In this report, we have prepared giant unilamellar vesicles (GUVs) of lipid membranes in the lo phase and investigated their physical properties using phase-contrast microscopy and fluorescence microscopy. GUVs of dipalmitoyl-phosphatidylcholine (DPPC)/cholesterol membranes and also GUVs of sphingomyelin (SM)/cholesterol membranes in the lo phase in water were formed at 20-37 degrees C successfully, when these membranes contained >/=30 mol % cholesterol. The diameters of GUVs of DPPC/cholesterol and SM/cholesterol membranes did not change from 50 to 28 degrees C, supporting that the membranes of these GUVs were in the lo phase. To elucidate the interaction of a substance with a long hydrocarbon chain with the lo phase membrane, we investigated the interaction of low concentrations (less than critical micelle concentration) of lysophosphatidylcholine (lyso-PC) with DPPC/cholesterol GUVs and SM/cholesterol GUVs in the lo phase. We found that lyso-PC induced several shape changes and vesicle fission of these GUVs above their threshold concentrations in water. The analysis of these shape changes indicates that lyso-PC can be partitioned into the external monolayer in the lo phase of the GUV from the aqueous solution. Threshold concentrations of lyso-PC in water to induce the shape changes and vesicle fission increased greatly with a decrease in chain length of lyso-PC. Thermodynamic analysis of this result indicates that shape changes and vesicle fission occur at threshold concentrations of lyso-PC in the membrane. These new findings on GUVs of the lo phase membranes indicate that substances with a long hydrocarbon chain such as lyso-PC can enter into the lo phase membrane and also the raft in the cell membrane. We have also proposed a mechanism for the lyso-PC-induced vesicle fission of GUVs.
When cells of a marine pseudomonad were washed with a solution consisting of 0.3 m NaCl, 0.05 m MgSO(4), and 0.01 m KCl (complete salts), they maintained their normal morphology. When washed with a solution of 0.05 m MgSO(4), they became plasmolyzed as indicated by both phase and electron microscopy. Suspensions of cells washed with 0.05 m MgSO(4) showed an increase in optical density (OD) when 0.3 m NaCl was added, and this was followed by a decrease in OD upon the further addition of 0.01 m KCl. Salts of other monovalent cations were not effective in replacing K(+) in producing the OD decrease. Phase-contrast microscopy revealed that the increase in OD was accompanied by a decrease in cell size, and the decrease in OD, by an increase in the cell size. Both phase and electron microscopy showed that the K(+)-dependent decrease in OD was accompanied by deplasmolysis of the cells. Na(+) was required in the suspending medium in addition to K(+) to obtain deplasmolysis. The intracellular K(+) concentration in cells which had been washed with complete salts and which had retained their normal morphology was found to be 0.290 m. In cells plasmolyzed by washing with 0.05 m MgSO(4), the intracellular K(+) concentration was 0.004 m. Deplasmolyzed cells contained 0.330 m K(+). The membrane profile of plasmolyzed cells was retained when protoplasts were formed. The protoplasts became spherical if incubated in a solution permitting the deplasmolysis of the parent cells. The evidence obtained indicates that plasmolysis and deplasmolysis under the conditions described was due to the loss and gain, respectively, of K(+) by the cells. The effect of Na(+) could be ascribed to its capacity to control the porosity of the cytoplasmic membrane of this organism.
We describe the histopathologic results of extracapsular lens extraction and silicone and poly(methyl methacrylate) (PMMA) intraocular lens (IOL) implantation in 36 rabbit eyes. Phase-contrast microscopy was used to examine precipitates on IOL surfaces and posterior capsules. Semithin and ultrathin sections were taken from the central cornea, anterior uvea, capsular bag, and retina near the posterior pole. The follow-up was one to 16 weeks. Silicone IOLs did not cause significantly less precipitation than PMMA IOLs. Precipitates consisted of spindle-shaped fibroblast-like cells, various forms of inflammatory cells and multinucleated giant cells, single melanophages, and irregularly arranged birefringent collagen fibers. Corneal endothelial edema was slightly more prominent in PMMA IOL implanted eyes. Significant retinal edema in the posterior pole area was not observed with either of the two lens types. Severe precipitation in the form of large clusters of pigment cells and inflammatory reactions seemed to depend on mechanical trauma (iris capture and lens dislocation) and individual animal reactions, but not on the lens type used.
Spores of Dictyostelium discoideum may be quantitatively activated with a heat treatment of 45 degrees C for 30 min. Heat activation at either higher temperatures of for longer duration at 45 degrees C resulted in damaged spores. The spores showed an increased postactivation lag time at 23 degrees C and an increased inability to respond to deactivation with 0.2 M sucrose. As the severity of supraoptimal heating increased, a greater percentage of the spores appeared to contain phase dark lesions and to lose viability. Oxygen uptake began to decrease during and after the appearance of the lesions. Using electron microscopy, the phase dark lesions were found to be mitochondria with disrupted cristae.
A technique is described by which unstained metaphases may be observed using phase contrast microscopy. The cells are fixed on a cover slip which then is turned over and onto a slide, leaving a thin air layer sandwiched between. Observation in phase contrast shows metaphases comparable to those observed after Giemsa staining.
The poxvirus strains NYVAC and MVA are two candidate vectors for the development of vaccines against a broad spectrum of diseases. Although these attenuated virus strains have proven to be safe in animals and humans, little is known about their comparative behavior in vitro. In contrast with MVA, NYVAC infection triggers greater cytopathic effect in a range of permissive and nonpermissive cell lines. The yields of NYVAC cell-associated virus in permissive cells (BHK-21) were slightly reduced compared with those of MVA infection. During the course of infection in HeLa cells, there is a translational block induced by NYVAC late in infection, which correlated with a marked increase in phosphorylation levels of the initiation factor eIF-2alpha. In contrast to MVA, the synthesis of certain late viral proteins was only blocked in NYVAC-infected HeLa cells. Electron microscopy (EM) analysis revealed that morphogenesis of NYVAC in HeLa cells was blocked at the stage of formation of immature viral forms. Phase-contrast microscopy, EM, flow cytometry, and rRNA analyses demonstrated that contrary to MVA, NYVAC infection induces potent apoptosis, a phenomenon dependent on activation of caspases and RNase L. Apoptosis induced by NYVAC was prevented when the virus gene C7L was placed back into the NYVAC genome, recovering the ability of NYVAC to replicate in HeLa cells and maintaining the attenuated phenotype in mice. Overall, our findings demonstrate distinct behavior between NYVAC and MVA strains in cultured cells, as well as a new role for the C7L viral gene as an inhibitor of apoptosis in NYVAC infection.
AIMS: (1) To make precise measurements and comparisons of various aspects of motility of three gastrointestinal pathogens, Helicobacter pylori, Campylobacter jejuni, and Escherichia coli, in log phase growth; (2) to provide background information on motility data to study the influence of pH, viscosity, and chemotactic factors, thereby gaining a better understanding of bacterial pathogenesis. METHODS: Computer image processing technology and phase contrast microscopy (Hobson BacTracker) were used to measure several indices of bacterial motility in real time. Ten clinical isolates of each species in log phase liquid culture were studied. RESULTS: C jejuni moved fastest, with a median curvilinear velocity (CLV) of 38.76 microns/s (range 29.08 to 52.82). Next was H pylori, median CLV 25.02 microns/s (range 12.07 to 29.07). E coli was the slowest, median CLV 12.73 microns/s (range 8.20 to 18.04). The straight line velocities showed similar trends. Measurement of track linearity (TL) showed that C jejuni moved the straightest (TL 60.3%), H pylori moved in wide circles (TL 28.7%), and E coli showed spinning movement without much linear displacement (TL 18.3%). There were significant differences in these three variables between the species studied, but no significant differences in measurements of time and frequency of halts between movement runs. CONCLUSIONS: The BacTracker provides a useful technical aid for measuring several indices of bacterial motility objectively, reproducibly, and precisely, which is difficult to achieve without computer assistance. Accurate quantification of motility provides a basis for studying the factors which influence bacterial motility. It can provide phenotypic measurements of the effect of flagellar gene depletion.
The nucleoid structure and the partition in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 were observed by a combination of phase-contrast microscopy and fluorescence microscopy. The nucleoids occurred as rounded fluorescent foci centrally located in the cells and as differences in fluorescence intensity between exponential and stationary phases. The cellular space occupied by the nucleoid in the stationary phase was larger than that in the exponential phase. Various shapes of nucleoid in the exponential-phase cells were observed, indicating that nucleoid separation was processed under cell cycle control. The number of cells which showed distinctive division stages was counted and the proportions of dividing cells were determined. About half of the observed cells were in the replication stage. More than 40% of the counted cells possessed a fully replicated but not separated form of nucleoid. Only 8% of the total cells clearly showed visible constriction. These results suggested that the post-replication period before cell division was relatively as long as the eucaryal gap period (G2); however, the period of visible cell constriction was almost the same as that of the bacteria.
Electron microscopy of intact tissue and cultured smooth muscle suggested the occurrence of phagocytosis of necrotic cells by normal vascular smooth muscle cells. Phagocytosis was further studied in vitro in muscle cells from guinea pig aorta, guinea pig vas deferens, and striated muscle from chick embryo by light and electron microscopy. The uptake of yeast cells (approximately 2-mum. diameter) into living cultured smooth muscle cells from guinea pig aorta was observed with phase contrast microscopy and differential interference (Nomarski) optics. Periodic acid-Schiff staining showed the yeast cells lined up in the cytoplasm of the muscle cells. Electron microscopy confirmed the uptake of yeast cells by differentiated muscle cells. Some of the yeast in the smooth muscle cells showed signs of disintegration suggesting digestion within the phagosome. Spontaneously contracting cultured smooth, cardiac and skeletal muscle cells incubated with latex spheres (0.3-mum. diameter) showed uptake and sequestration of the spheres. The ability of muscle cells to phagocytose necrotic cells and other particulate matter may be important in atherogenesis and in the repair of tissue after injury.
Effects of macrophage-derived growth factor on the progression of the cell cycle of cultured aortic smooth muscle cells were investigated by using rabbit peritoneal macrophage conditioned-medium (M phi-CM) as the source of the growth factors. DNA content of individual SMC in the cell population which had been exposed either to platelet-poor plasma serum (PPPS) or to M phi-CM was also measured by flow cytometry and microspectrophotometry, as well as by phase contrast microscopy. The results showed that the cells quiescent in PPPS medium had a high population not only in G0/G1 but also in G2 phase. This indicated that the cell cycle process was blocked both in G0/G1 and G2 phase. The cell cycle distribution of SMCs exposed to M phi-CM for 48 hours was very similar to that cultured in 10% fetal calf serum medium. It is suggested that M phi-CM can promote the cell growth-arrested in ppps medium passing through G0/G1 into S phase as well as through G2 into M phase.