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PubMed1974

Germination of yeast spores lacking mitochondrial deoxyribonucleic acid.

A population of petite ascospores (mitochondrial deoxyribonucleic acid [mtDNA]-less), produced by brief ethidium bromide (EthBr) mutagenesis prior to transfer to sporulation medium, was used to examine the role of the mitochondrial genetic system on germination and outgrowth in Saccharomyces cerevisiae. Petite ascospores, which are morphologically indistinguishable by phase-contrast microscopy from wild-type spores, germinate and proceed through outgrowth at a rate and extent only slightly less than that of wild-type spores. Both developmental processes occurred in the absence of mtDNA synthesis and measurable cytochrome oxidase activity. These results indicate that neither respiration nor a functional mitochondrial genome are required for germination and outgrowth. The properties of the petite clones were typical of petites formed during vegetative growth. Individual sporal clones differed markedly from each other in suppressiveness. Petite sporal clones which exhibited a high degree of supressiveness also contained a reduced but detectable amount of mtDNA of altered buoyant density. One clone contained a unique mtDNA with a buoyant density higher than that of wild-type mtDNA.

Centrifugation, Density Gradient↗
PubMed1974

Effect of polyoxin D on chitin synthesis and septum formation in Saccharomyces cerevisiae.

The normal sequence of cell separation in Saccharomyces cerevisiae begins with the formation of a primary septum, presumably consisting of chitin, on which secondary septa are later deposited. In the presence of the antibiotic polyoxin D, a potent inhibitor of chitin synthetase, pairs of abnormal cells of two different types were observed by phase-contrast microscopy: the "exploded pair," consisting of two lysed cells from which the cytoplasm had been extruded at the cell junction, and the "refringent pair," consisting of two highly refractile cells joined by a thin bridge. Thus, in both cases the septal region appears to be affected. Observations with the electron microscope showed that the primary chitin septum was not formed in either of these cell types, and as a consequence secondary septa of varying thicknesses were laid down in an abnormal pattern. With [(3)H]glucose as carbon source the incorporation of tritium into the chitin of abnormal cells was inhibited about 90%, whereas the labeling of mannan was normal and that of glucan somewhat reduced. The effective concentrations of polyoxin D (0.1 to 1 mg/ml) were much greater than those required to inhibit chitin synthesis in vitro. Dimethylsulfoxide and amphotericin B, both known to increase cell permeability, enhanced the action of the antibiotic.

Anti-Bacterial Agents↗
PubMed1975

Polarized relationship of bacterial spore loci to the "old" and "new" ends of sporangia.

The frequency of association of spore loci with the "old" and "new" ends of rod-shaped sporangia in batch cultures of Bacillus megaterium ATCC 19213 was estimated by phase contrast microscopy. The analysis was facilitated by (i) the association of most of the sporangia into chains of two to five sporangia and (ii) the occurrence of two types of cross wall distinguishable by their degree of splitting. It was concluded that a newly formed spore is located at the "old" end of a sporangium. By inference, the sporulation division septum locus is distal to the ultimate normal cell division septum, i.e., proximal to the "old" pole of the B. megaterium sporangium. This result is discussed in relation to deoxyribonucleic acid segregation during sporulation.

Bacillus megaterium↗
PubMed1984

Phase separation between nucleoid and cytoplasm in Escherichia coli as defined by immersive refractometry.

The refractive indices of nucleoid and cytoplasm in Escherichia coli were derived theoretically and experimentally. For the theoretical estimates, we made use of the known macromolecular composition of E. coli B/r (G. Churchward and H. Bremer, J. Theor. Biol. 94:651-670, 1982) and of estimates of cell and nucleoid volumes. These were obtained from micrographs of living bacteria made with a confocal scanning light microscope. The theoretical values were calculated, assuming that all DNA occurred in the nucleoid and that all protein and RNA occurred in the cytoplasm. Comparison with experimental refractive index values directly obtained by immersive refractometry showed that, besides its DNA, the nucleoid must contain an additional amount of solids equivalent to 8.6% (wt/vol) protein. With the nucleoid containing 6.8% (wt/vol) DNA and 8.6% (wt/vol) protein and the cytoplasm containing 21% (wt/vol) protein and 4% (wt/vol) RNA, a mass difference is obtained, which accounts for the phase separation observed between the nucleoid and cytoplasm in living cells by phase-contrast microscopy. The decrease in the refractive index of the nucleoid relative to that of the cytoplasm observed upon, for instance, OsO4 fixation was interpreted as being indicative of the loss of protein content in the nucleoid.

Chloramphenicol↗
PubMed1961

Effects of some mechanical factors on the endospores of Bacillus subtilis.

Knaysi, Georges (Cornell University, Ithaca, N. Y.) and Harold R. Curran. Effects of some mechanical factors on the endospores of Bacillus subtilis. J. Bacteriol. 82:691-694. 1961.-Phase contrast microscopy was used to study incipient spore germination and early growth under conditions involving agitation with a platinum wire loop, sonic oscillation, and freezing and thawing. Treatment resulted in change in optical properties and destruction of a certain proportion of the spores. Up to one-tenth of the spores was destroyed by loop agitation. Sonic oscillation had little apparent effect upon incipient germination (darkening) of the spores, but subsequent development involving growth was stimulated in a considerable proportion. The effect of freezing and thawing upon incipient germination is discussed.

Bacillus subtilis↗
PubMed1967

Fine structure of Sarcina maxima and Sarcina ventriculi.

The fine structure of Sarcina maxima and S. ventriculi was studied by electron and phase-contrast microscopy. The two organisms differ mainly with respect to their cell surface. A thick cellulose layer present on the cell wall of S. ventriculi was not observed on the surface of S. maxima. Carbon replication indicated that the outer surface of S. ventriculi is rough in contour, probably as the result of the fibrillar nature of the accumulated cellulose. The cytoplasm of both sarcinae contains inclusions similar to polysaccharide and polymetaphosphate granules. Mesosomes were observed in cells of S. maxima. Packets of S. ventriculi generally comprise a larger number of cells and are more irregularly constructed than those of S. maxima. Cells in large packets of S. ventriculi assume flattened or otherwise irregular shapes, whereas cells of S. maxima maintain a more uniform appearance.

Microscopy, Electron↗
PubMed1967

Permeability properties of Rickettsia mooseri.

The passive permeability properties of Rickettsia mooseri to both inorganic and organic solutes have been examined. Visual observations by phase-contrast microscopy of rickettsiae in macerated yolk sacs taken directly from heavily infected eggs revealed plasmolysis with hypertonic NaCl and KCl as well as with sucrose solutions. In contrast, similar visual studies of rickettsiae which had been subjected to freezing or to a purification process, or both, were plasmolyzed by hypertonic sucrose but not by hypertonic NaCl and KCl. These primary observations were extended to a variety of solutes and were placed on a quantitative basis by use of optical density and radioisotope dilution methods. Intracellular Na(+) and K(+) concentrations in processed rickettsiae, measured by flame photometry, closely paralleled the concentration of these ions in the suspending medium. It was concluded that R. mooseri appears to possess an osmotically active, functional, and structural membrane distinct from the cell wall, located at the surface of a structure analogous to the bacterial protoplast. In the intact organism, this membrane is passively impermeable to sucrose, NaCl, and KCl. However, altered permeability properties, especially to inorganic electrolytes, may be expected in rickettsiae which have been stored in the frozen state and subjected to a lengthy purification process.

Animals↗
PubMed1968

Surface structure of yeast protoplasts.

The fine structure of the yeast cell wall during protoplast formation was studied by means of phase-contrast microscopy and the freeze-etching technique. The freeze-etching results indicated that at least in some cases the entire wall substance was not removed from the surface of the protoplasts. After a treatment of 30 min to 3 hr with 2% snail enzymes, an innermost thin wall layer as well as remnants of the fibrillar middle layer sometimes could be demonstrated.

Cell Membrane↗
PubMed1986

Techniques for examining Pneumocystis carinii in fresh specimens.

Pneumocystis carinii was examined in fresh preparations of infected rat lung homogenates and tissue culture supernatants by a variety of light microscope techniques, vital dyes, and histologic stains. Phase-contrast microscopy, Nomarski interference-contrast microscopy, and bright-field microscopy with oblique illumination provided excellent views of P. carinii. Erythrosin B, and to a lesser extent trypan blue, were helpful in assessing organism viability. The use of Triton X-100-Giemsa stain permitted differentiation of the developmental stages in the P. carinii life cycle. The techniques developed here are easily adaptable to the microbiology laboratory and thus should have important clinical and research applications.

Animals↗
PubMed1974

Effects of actinomycin D on the cytopathology induced by poliovirus in HEp-2 cells.

One possible mechanism of virus-induced cell damage is that the redistributed (released) lysosomal enzymes produce the cytopathic effect during cytolytic types of infections such as poliovirus in HEp-2 cells. To determine if the lysosomal enzyme redistribution and cell damage are host-cell directed, we studied sensitivity of these events to the action of actinomycin D. By the use of actinomycin D at concentrations producing the least toxicity but maximal effectiveness in shuting down cell RNA synthesis, it was shown that the cytopathic effect and enzyme redistribution were not inhibited and, therefore, not directly controlled and induced by the cell genome in response to the virus infection. Evaluation of cytopathic effect by a phase contrast microscopy method detected changes earlier than the erythrocin B uptake method.

Carcinoma, Squamous Cell↗
PubMed2004

T lymphocyte mediated lysis of mitomycin C treated Tenon's capsule fibroblasts.

AIMS: To evaluate the effect of T cell co-culture on mitomycin C treated and untreated Tenon's capsule fibroblasts. METHODS: IL-2 dependent allogeneic T cells were incubated over a monolayer of mitomycin C treated or control fibroblasts. Fibroblast numbers were evaluated by direct counts using phase contrast microscopy. To determine whether T cell mediated lysis was a consequence of MHC mismatch, co-culture experiments were repeated with autologous T cells. The effect of Fas receptor blockade was established by co-incubation with a Fas blocking (M3) antibody. RESULTS: T cell co-culture resulted in a dramatic reduction in fibroblast survival compared to mitomycin C treatment alone (p = 0.032). T cell killing required fibroblast/lymphocyte cell to cell contact and was observed in both allogeneic and autologous co-culture experiments. Fas blocking antibodies did not significantly inhibit T cell killing (p = 0.39). CONCLUSION: T cells augment mitomycin C treated fibroblast death in vitro. Similar mechanisms may contribute to the cytotoxic effect of mitomycin C in vivo and account for the largely hypocellular drainage blebs that are observed clinically.

Antibiotics, Antineoplastic↗
PubMed2006

Mechanical energy from intraocular instruments cause emulsification of silicone oil.

AIM: The complications of intraocular silicone oil include emulsification, which may lead to vision-threatening disorders such as keratopathy, secondary glaucoma and retinopathy. The authors study the effect of mechanical energy from surgical instruments on the emulsification of silicone oil. METHODS: Three different handpieces (phacofragmentation, phacoemulsification, high-speed vitrectomy) were placed at the interface of balanced salt solution and silicone oil (1000 and 5000 cst, 200 fluid; Dow Corning). The phacofragmentation handpiece was evaluated over different ultrasound powers and duration. Emulsification was quantified with phase-contrast microscopy and manual counting of digital photographs by a masked examiner. In addition, phacoemulsification was performed in the anterior chamber of a human ex vivo eye with full-fill silicone oil. RESULTS: Emulsification increases with higher phacofragmentation power and duration and is greater for 1000 cst silicone oil. Emulsification of silicone oil occurs with phacoemulsification and high-speed vitrectomy handpieces. CONCLUSIONS: The energy from surgical handpieces is sufficient to result in silicone oil emulsification.

Emulsions↗
PubMed1965

PREPARATION AND USE OF FIXATIVE SLIDES FOR THE DIAGNOSIS OF TRICHOMONAS VAGINALIS.

Clean microscopic slides are dipped in a solution of mercuric chloride and sucrose in 30% alcohol and allowed to dry. A thin transparent film of cytological fixative is thus formed on both sides of the slide. Fixative slides, prepared in this manner, can be stored indefinitely provided that they are not exposed to a warm, humid atmosphere. When a drop of fluid containing bacteria, protozoa, or mammalian cells is spread over the surface of such a slide the formed elements are immediately fixed, the finer cytological details being well preserved. Staining and examination are carried out when the specimen arrives at the laboratory. This method is particularly suitable for a diagnostic laboratory service in which most of the specimens arrive by mail. The method has been successfully employed in the diagnosis of Trichomonas vaginalis infection of the female genital tract. This parasite was found by phase-contrast microscopy in 47% of 1,000 women examined at a police court clinic. Fixative slide preparations were also obtained from these women at the time of their medical examination. Of these specimens, 38.6% showed the presence of Tr. vaginalis, and 10% were also found to be infected with N. gonorrhoeae. The fixative slide technique may also prove of value in such widely separated fields as exfoliative cytology and the diagnosis of primary syphilis.

Animals↗
PubMed1972

An evaluation of the Celloscope 401 electronic blood cell counter.

For counting erythrocytes the instrument was precise, with a mean coefficient of variation of 1.21%. Erythrocyte counts showed close agreement with results obtained on a Coulter A electronic counter of proven accuracy. When the Celloscope 401 was modified by the manufacturers to eliminate electrical interference from other laboratory equipment, satisfactory precision and accuracy for white cell counting was obtained. Using cetrimide diluent the coefficient of variation was 1.6% but when using saponin/saline diluent the coefficient of variation was 3.5%. For leucocyte counting there was close agreement between duplicate tests performed on the Celloscope 401 and the Coulter S. The instrument was capable of satisfactory precision and accuracy in platelet counting, provided that the sedimentation method was used to obtain a platelet-rich plasma. The best results were obtained if a two-step dilution was carried out with a first dilution in 10% EDTA and a second in 2.5 mM cocaine in water. Using this method the precision study indicated a coefficient of variation of 3.11%. Close agreement was obtained between platelet counts on the Celloscope 401 when compared with the results obtained either by phase-contrast microscopy or using another electronic counter. Allowing for predilution and duplicate counts on each sample, the rate of throughput was approximately 32 samples per hour. Throughout the test period, the instrument remained electronically and mechanically stable.

Blood Cell Count↗
PubMed1973

The optical and electron microscopic determination of pulmonary asbestos fibre concentration and its relation to the human pathological reaction.

The quantitative extraction of asbestos fibres from asbestotic lung by alkali digestion has been refined by maceration of the tissue without prior drying, the minimum use of centrifugation, and the adoption of phase contrast microscopy. Preliminary experiments suggested that, using this technique, asbestos fibre counts were accurate to within at least +/- 20% and in most instances to within +/- 10%. The method was used to assess asbestos concentrations in lung tissue showing various degrees and forms of fibrosis. The results, as determined by light microscopy, indicated that uncoated fibres generally outnumbered coated fibres. In mild and moderate asbestosis there was a progressive increase in concentration of asbestos fibres, both coated and uncoated, with increasing severity of fibrosis, whereas in severe asbestosis no correlation existed between the fibre concentration and the form or the extent of the pathological reaction. It is suggested that the severe fibrosis results from the supervention of non-specific inflammatory processes. Asbestos fibre diameter distributions, gauged by electron microscopy, were fairly constant irrespective of the degree of fibrosis. Optically visible fibres constituted between 12 and 30% of the total, so that an optical count may be said to give an approximate indication of the total asbestos concentration and, so far as asbestosis is concerned, may well serve for comparative purposes. The relation between asbestos and neoplasia will, however, require identification and quantitation of particular types of the mineral by microanalytical techniques.

Asbestos↗
PubMed1975

Ultrastructural effects of lysozymes on the cell wall of Caryophanon latum.

When Caryophanon latum was exposed to egg white lysozyme in isotonic sucrose and observed by phase-contrast microscopy, protoplasts emerged along the length of the trichomes, apparently at sites corresponding to cross septa. Electron microscopy of sections revealed that this enzyme initially attacked the core of the septal peptidoglycan and delamination of septa resulted. The inner densely staining layer of the lateral and polar wall (considered to contain peptidoglycan as the major component) remained intact except for destruction at the advancing tip of partial septa; protoplasts or cell debris could escape from the gaps formed at developing septa. Treatment of intact trichomes with pronase, a lipase - phospholipase C mixture, EDTA, glutaraldehyde, or heat, before exposure to egg white lysozyme did not alter this pattern nor did it render the remaining peptidoglycan more susceptible to attack. The wall material external to the peptidoglycan was solubilized by pronase. The peptidoglycan remaining after lysozyme treatment was not morphologically changed by treatment with pronase. Lysozyme derived from Chalaropsis hydrolyzed incomplete septa initially, while the lateral and polar wall and complete septa were degraded later. Therefore, it is most probable that the inner dense layer does contain the peptidoglycan component and that some biochemical maturation distinguishes the substrate for these enzymes in the lateral wall and septa.

Bacteria↗
PubMedDate not supplied

Validation of the morphologic end point of necrosis in rat hepatocytes subjected to oxyradical damage.

We have used phase-contrast microscopy to determine a necrotic end point of the order of minutes in primary hepatocytes exposed to oxyradicals generated with xanthine oxidase plus hypoxanthine. This study examines whether the morphologic end point thus determined agrees with other criteria of cell necrosis. When 95-100% of the cells were shown to be necrotic by our morphologic assay, transmission electron microscopy confirmed definitive subcellular evidence of cell death, trypan blue exclusion revealed a 92% loss in the ability of cells to exclude the dye, and there was a 47% specific release of 51Cr (versus a 50% theoretical value). In contrast, the appearance of extracellular aspartate aminotransferase activity was relatively slow and did not corroborate the morphologic end point. In summary, we have validated the morphologic end point in our cell-based assay of oxyradical damage.

Animals↗
PubMed1980

Kinetics of ADP-induced human platelet shape change: apparent positive cooperativity.

The kinetics of ADP-induced human platelet shape change have been examined. Initial velocities of platelet shape change were estimated by two methods: (1) the slope of the initial decrease in light transmission through stirred, citrated platelet-rich plasma, and (2) direct examination of platelet morphologies by phase-contrast microscopy. In both cases, a value of the Hill coefficient, NH, significantly greater than 1 is obtained (2.0 +/- 0.2 and 1.8 +/- 0.2, respectively). The observed elevated value of NH is not due to a substantial fraction of the ADP being platelet bound, the presence of factors in the plasma, platelet heterogeneity, or the influence of the rate of platelet shape change reversion. Our observations suggest that ADP-induced platelet shape change may be a positively cooperative or "threshold" type response.

Adenosine Diphosphate↗