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PubMed1977

Comparison of the effects of mecillinam and 6-aminopenicillanic acid on Proteus mirabilis, Escherichia coli, and Staphylococcus aureus.

Single strains of Proteus mirabilis, Escherichia coli, and Staphylococcus aureus were grown on filter membranes placed on agar containing concentration series of mecillinam (FL 1060), 6-aminopenicillanic acid (6-APA), or ampicillin. P. mirabilis and E. coli were also exposed to combinations of mecillinam or 6-APA with ampicillin. Colony-forming units were counted, and cells were examined by interference phase-contrast and transmission electron microscopy. Mecillinam and 6-APA were very effective in reducing the viability of the two gram-negative species, but they were less effective against S. aureus. Combinations of mecillinam and 6-APA with ampicillin acted synergistically against the gram-negative bacilli. When the antibiotics were presented consecutively, their effects on viability were usually no greater than the effects of the individual antibiotics acting alone. When P. mirabilis and E. coli were exposed to mecillinam alone or in combination with ampicillin, the cells became rounded. 6-APA alone or in combination with ampicillin produced elongated polymorphic cells in these species. The most unusual morphological effects were ultrastructural. Mecillinam and, to a lesser extent, 6-APA produced inward growth of numerous pairs of trilamellar membranous structures within the cells. It is possible that these membranes represent the growth initiation of aberrant cross walls. Both mecillinam and 6-APA produced multiple, thick cross walls in S. aureus.

Ampicillin↗
PubMed1976

Bacteriological study on effects of beta-lactam group antibiotics in high concentrations.

The growth and viability of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa exposed to various concentrations of a number of beta-lactam group antibiotics were determined. In S. aureus, the bacteriolytic and bactericidal activity of these drugs was lower at very high drug concentrations than that occurring at low concentrations, but these phenomena were not observed in E. coli and P. aeruginosa. Under phase-contrast and scanning electron microscopy, S. aureus treated with high concentrations of beta-lactam group antibiotics revealed a lower frequency of bacteriolysis than at low drug concentrations, and similarly by transmission electron microscopy fewer cells were transformed into spheroplasts at high drug concentrations. However, swelling of the cell wall septum was seen in many cells. Spheroplast formation occurred with the highest frequency at drug levels near the minimum inhibitory concentration and became less frequent as drug concentrations were increased.

Anti-Bacterial Agents↗
PubMed1978

Cytopathogenicity of Mycoplasma hyopneumoniae in porcine tracheal ring and lung explant organ cultures alone and in combination with monolayer cultures of fetal lung fibroblasts.

Porcine tracheal rings and lung explants alone and in combination with monolayer cultures of porcine lung fibroblasts (PLF) were separately inoculated with virulent strains of Mycoplasma hyopneumoniae and incubated at various times. The preparations were observed by bright-field, phase-contrast, and scanning electron microscopy. In PLF cultures, the strains at initial concentrations of 10(1.3) colony-forming units/ml increased within 3 days to 10(6) colony-forming units/ml, showed progressive clustering on the cells, and caused some sloughing. Introduction of a tracheal ring or lung explant into these mycoplasma-infected PLF cultures caused the explant to lose its epithelial ciliary motility. Eventually parts or whole cells of the respective ciliated epithelium were lost. Without infected PLF monolayers, the explants inoculated with M. hyopneumoniae were less susceptible to infection. When uninfected explants were incubated for 18 days or kept in stock for 2 months, they did not show the above changes. With 5 h postinoculation, M. hyopneumoniae cultures became intimately associated with the PLF culture, but when epithelial cell sloughing occurred, the mycoplasmal cells became dependent on the introduction of a fresh PLF monolayer or a tracheal or lung explant for survival.

Culture Techniques↗
PubMed1977

Morphology and ultrastructure of Crenothrix polyspora Cohn.

Naturally grown cell material of Crenothrix polyspora from the well of a waterworks was studied by means of phase-contrast and Nomarski interference microscopy as well as by transmission electron microscopy. The material consisted of clusters of sheathed filaments up to 2 cm long. Propagation forms observed were nonmotile, spherical cells that arose by simple ("macrogonidia") or multiple ("microgonidia") septation of the filamental tips. Ultrastructural analysis revealed Crenothrix to be procaryotic and gram negative, with several layers of sheath material surrounding the filaments. On thin sections, individual cells had elaborate membrane systems in the form of lamellar stacks. They resembled thylakoids of photosynthetic bacteria. Spectrophotometric analysis gave no indication of photosynthetic pigments. The cells also contained large hexagonal bodies, rod-shaped fibrillar elements, and polyphosphate granules.

Bacteria↗
PubMed1977

Effect of naturally occurring apatites on growth and morphology of algae.

Crystals (30--100 micrometer) of selected naturally occurring apatite (Ca10 (PO4)6(OH, F)2) samples were added to P-free (less than 0.001 microgram/ml total P) Bristol's medium (1-1000 microgram/ml of apatite) as the sole source of ortho-PO43-. The media were inoculated with washed, non-axenic cells of three chlorophycean algal species cultivated under PO43--deficient conditions. Phase-contrast and scanning electron microscopy revealed that at low slurry densities (1-10 microgram/ml of apatite), Ankistrodesmus braunii (ATCC 2744) cells were morphologically distorted. At concentrations of 100 and 1000 microgram/ml of apatite, more than 85% of the cells had undergone autospore formation within 7--10 days of incubation at 20 degrees C. Most autospores formed failed to germinate under high nutrient conditions. Scenedesmus longus (No. 1236) formed colonies when cultivated in Bristol's medium but daughter cells displayed a Chodatella-like unicellular morphology when grown in apatite media. Test algal species (Chlamydomonas dysosmos, S. longus, A. braunii) showed a marked preference for growth on apatite crystals over non-nutritive surfaces. Unialgal and mixed-algal cultures produced an extensive matrix of extracellular fibrous material in response to growth on crystals at concentrations greater than 10 microgram/ml of apatite.

Apatites↗
PubMed1979

Marginal bands in camel erythrocytes.

The elliptical, anucleate erythrocytes of camels have been examined for the presence of marginal bands and their constituent microtubules. Lysis of erythrocytes under microtubule-stabilizing conditions readily revealed marginal bands in at least 3 % of the cells, as observed by phase-contrast and darkfield light microscopy. Microtubules plus a marginal band-encompassing network of material are visible in lysed cell whole mounts with transmission electron microscopy. Marginal band microtubules are also evident in electron micrographs of thin-sectioned camel erythrocytes identifiable as reticuloyctes on the basis of submaximal electron density (reduced haemoglobin iron content) and presence of polysomes. The results suggest that marginal bands may be involved in morphogenesis of camel erythrocytes but are not required for maintenance of their ellipticity after cells are fully differentiated.

Animals↗
PubMedDate not supplied

[Light microscopic and electron microscopic study of the cells of tissue cultures irradiated with a neodymium laser].

Damage of tissue culture cells (Hela, a human amnion, a primary rat liver culture) caused by the neodymium laser radiation was studied by time-lapse phase-contrast microfilming and electron microscopy. The tested cultures were shown to display different sensitivity and the degree of cell alteration within the same cultures varied considerably. A physical mechanism of cell damage is probably of thermal and shock-wave nature.

Amnion↗
PubMed1992

Total internal reflection fluorescence microscopy (TIRFM). II. Topographical mapping of relative cell/substratum separation distances.

A simplified model of TIRF optics was used to quantitate the relative membrane/substratum separation distances from the spatial pattern of TIRF image brightness. Phase-contrast and total internal reflection fluorescence microscopy (TIRFM) images were collected of bovine aortic endothelial cells (BAEC) plated onto glass microscope slides for 15 min, 30 min and 24 h. BAEC adherent for 15 min showed an absence of a focal contact morphology, with the region of closest apposition beneath the cell center. After 30 min, multiple contacts with the surface were established and the morphology became more irregular. BAEC attached for 24 h showed well-defined focal contact regions aligned in characteristically striated patterns. The relative distance between closest and farthest membrane/substratum separations are consistent with reported distance between focal and matrix contacts. Topographical maps of membrane/substratum separation distances over the entire ventral surface of the plated cells were constructed to demonstrate the utility of quantitative TIRF microscopy.

Animals↗
PubMed1992

Enhancing in vitro fertilization of mouse oocytes by partial zona pellucida digestion.

This work was undertaken in order to evaluate the effect of partial zona digestion on fertilization in vitro of mouse oocytes and assess zona surface changes induced by the procedure. Three hundred forty-six oocytes allocated for treatment were exposed to Ham's F-10 medium supplemented with 0.5% Pronase for either 3 min (188 oocytes) or 5 min (158 oocytes); 324 oocytes served as controls. Oocyte losses incurred as a result of the procedure were small (15 oocytes; 4.3%). Control and Pronase-treated oocytes were each divided into four subgroups and inseminated with 5 x 10(5), 5 x 10(4), 5 x 10(3), or 5 x 10(2) sperm cells/ml. Fertilization was assessed 8 hr following insemination by the appearance of two pronuclei and development to the two- to four-cell stage the following day. The morphology of the zona pellucida following Pronase treatment was assessed by phase-contrast and scanning electron (SEM) microscopies performed immediately after treatment. Fertilization rate of control oocytes was 80% at a sperm concentration of 500,000/ml and gradually declined to approximately 30% at 500 cells/ml. In contrast, treated oocytes inseminated with 500 sperm cells/ml demonstrated a normal rate of fertilization. At this low sperm concentration the longer Pronase treatment was significantly (P less than 0.05) more efficient in enhancing fertilization (69 and 88% for 3 and 5 min of Pronase treatment, respectively). Polyspermic fertilization was not observed in any of the subgroups. Phase-contrast microscopic examination of oocytes at the time of Pronase treatment showed an initial swelling of the zona pellucida for 30-60 sec with a time-dependent increase in its transparency.

Animals↗
PubMed1975

Tropomyosin antibody: the specific localization of tropomyosin in nonmuscle cells.

An antibody against purified chicken skeletal muscle tropomyosin is used in indirect immunofluorescence to visualize the localization of tropomyosin in a variety of nonmuscle cells. The antibody produces a fluorescent pattern which is very similar to that obtained with an actin-specific antibody. This pattern is composed of fluorescent fibers which are shown to be coincident with the fibers seen with phase-contrast optics. High resolution epifluorescent microscopy reveals that fibers stained with the actin antibody show a continuous fluorescence, while fibers reacted with the tropomyosin antibody show a periodic fluorescence. Measurements indicate that the lengths of the fluorescent segments are variable with an average of 1.2 mum while the spacing between segments is approximately 0.4 mum.

Actins↗
PubMed1992

Microinjection of smg/rap1/Krev-1 p21 into Swiss 3T3 cells induces DNA synthesis and morphological changes.

Microinjection of either Ki-rasVal-12 p21 or the GDP-bound form of Ki-ras p21 plus smg GDP dissociation stimulator (GDS), a stimulatory GDP/GTP exchange protein for Ki-ras p21, smg/rap1/Krev-1 p21, and rho p21, into quiescent Swiss 3T3 cells induced DNA synthesis irrespective of the presence or absence of insulin. The guanosine 5'-(3-O-thio)triphosphate (GTP gamma S)-bound form of smg p21B or the GDP-bound form of smg p21B plus smg GDS also induced DNA synthesis but only in the presence of insulin. Either the GDP-bound form of Ki-ras p21 or the same form of smg p21B alone was inactive, but smg GDS alone was slightly active only in the presence of insulin. The morphology of the cells was analyzed by scanning electron, phase-contrast, and confocal laser scanning microscopies. Ki-rasVal-12 p21 induced membrane ruffling irrespective of the presence or absence of insulin. The GTP gamma S-bound form of smg p21B showed the same effect only in the presence of insulin. Either the GDP-bound form of Ki-ras p21, the same form of smg p21B, or smg GDS alone was inactive. Upon microinjection of Ki-rasVal-12 p21, stress fibers markedly decreased and the cells became round and piled up. In contrast, upon microinjection of the GTP gamma S-bound form of smg p21B, stress fibers did not markedly decrease and the cells neither became round nor piled up. These results indicate that both ras p21 and smg p21 are mitogenic in Swiss 3T3 cells but that their actions are slightly different.

3T3 Cells↗
PubMed1992

Neural regeneration in the noise-damaged chinchilla cochlea.

Recent studies in the bird ear have shown that degenerated hair cells are sometimes replaced by regenerated receptor cells. The present study evaluated the adult mammalian cochlea for evidence of hair-cell and nerve-fiber regeneration. Eighty-eight noise-damaged chinchilla cochleas were examined as plastic-embedded whole mounts by phase-contrast and bright-field microscopy. No signs of hair-cell regeneration were found. However, 32 (70%) of 46 cochleas damaged by high-intensity noise and 20 (48%) of 42 cochleas damaged by moderate-intensity noise contained a variable number of nerve fibers which appeared to be regenerated. These fibers, which were located in severely damaged areas of organ of Corti, differed from residual fibers with respect to their diameters, the degree and pattern of myelination, and by the abnormal paths they followed within the osseous spiral lamina and on the basilar membrane. The number of regenerated fibers varied with type of exposure and length of recovery. The strongest response was found in ears exposed to a high-intensity, low-frequency noise. The results described here indicate that a potential exists for the biological restoration of the mammalian inner ear.

Animals↗
PubMed1992

[A method for studying the three-dimensional structure of organs and tissue in pathology].

A new technique of scanning electron microscopy for visualization of hidden cellular surfaces in organs and tissue has been developed. It employs the sample incubation in 30% potassium hydroxide and 96% alcohol mixed in equal amounts and dissociation until single cells appear in the solution, this being checked under phase-contrast microscope. The method is beneficial in non-standard cases of three-dimensional organization of organs and tissues.

Animals↗
PubMed1992

A novel structure associated with a lampbrush chromosome in the chicken, Gallus domesticus.

At a site near the end of the short arms of lampbrush bivalent 2 in the chicken (Gallus domesticus) there is always a marker structure that appears in the phase-contrast light microscope as a solid object with diffuse edges measuring about 4 microns across. When examined by transmission electron microscopy in thin section, this object appears as a loose bundle of fibres. In some preparations individual fibres appear 15-16 nm thick, smooth in outline and solid in cross-section. In other preparations they are 32-38 nm thick, rougher in outline and ring-like in cross-section. High-resolution scanning electron micrographs of the chromosome 2 marker show it to be a loose bundle of spaghetti-like fibres that is quite unlike anything previously seen on a lampbrush chromosome of any organism. As with the sectioned material, fibres in some preparations were smooth and 15-16 nm in diameter, whereas those in others were more knobbly and about 35 nm thick. The fibres appear to branch and in some cases it is clear that the daughter strands of a branch have the same dimensions as the parent strand. Free ends are rare. Total length of fibre material present at one marker locus is estimated to be between 500 and 2000 microns. Similar structures are not present on the lampbrush chromosomes of quail, wood pigeon or chaffinch. The nature of this fibrous marker, referred to in this paper as the "spaghetti marker", is discussed in relation to lampbrush chromosome function and to events that take place during the lampbrush phase of oogenesis in chicken. Evidence is discussed in relation to the possibility that the chromosome 2 marker represents a novel form of nuclear RNP or the specific association of some structural protein with one chromosome locus.

Animals↗
PubMed1992

Platelet plasma membrane is equally distributed between surface and osmotically-evaginable surface-connecting membrane, independent of size, subpopulation or species.

Osmotic swelling can double the external plasma membrane surface area of normal human platelets. The surface-connected open canalicular system (SCCS) has been proposed as the major source for this additional membrane. As bovine (B) platelets have been reported to lack SCCS, we compared osmotic swelling for B and human (H) cells. Addition of water to platelet-rich-plasma (10-90% v/v) caused sequential shape change and osmotic spherocyte (OS) formation, analyzed for size and surface area changes from time-dependent phase-contrast videomicroscopic images. Selected samples were fixed and stained with tannic acid prior to osmic acid fixation for visualization of open SCCS by transmission electron microscopy. Bovine platelets required 3-4x less water dilution of PRP than human platelets, with significant OS forming at 20% water addition. Continued water dilution converted 50% of platelets to OS, with maximally stable swelling and no significant lysis for bovine OS up to 60% dilution. Electron micrographs of unactivated discocytes (D) and of optimally-swollen OS showed open SCCS in human D not detectable in any of the swollen platelets, though granules, mitochondria and a small number of vesicles and vacuoles persisted. No evidence for any open SCCS was found for bovine D or OS, though the OS otherwise appeared similar to human OS. Geometric measurements of D and nonlysed OS showed a stable, maximal 2.1 +/- 0.1 fold increase in external plasma membrane surface area with osmotic swelling, identical for different-sized normal human platelets (mean volume V = 2.8-6.8 fl), for abnormally-large platelets (V = 8.7-11.6 fl), or for bovine or rabbit platelets (V = 3-4 fl). As osmotic swelling appears to primarily externalize SCCS in human platelets, the identical relative amounts of internal membrane externalized for bovine platelets is hypothesized to arise from an osmotically more labile, "closed", and structurally simpler SCCS or from a distinct membrane source than in human platelets. It appears that the surface to invaginated ("SCCS") plasma membrane is kept constant for human, bovine and rabbit platelets, independently of platelet production, size-dependent subpopulations, or of platelet ageing.

Animals↗
PubMed1992

Responses of the solitary olfactory receptor cell of the guinea pig.

Olfactory receptor cells were isolated from the nasal mucosa of the guinea pig, using enzymatic digestion and mechanical dissociation. Under a phase-contrast microscope, the cells can be readily distinguished from other tissues, because of morphological characteristics. The intracellular free calcium ion concentrations ([Ca2+]i) of these cells were determined using the Ca2+ sensitive dye fura-2 and digital imaging microscopy. In the presence of 1 microM ionomycin, calcium-ionophore, there was an irreversible increase in [Ca2+]i in all the cells. During prolonged stimulation by 10 mM n-Amyl acetate under standard conditions, 16 of the 40 isolated olfactory cells responded with [Ca2+]i. There was no evidence of any increase in [Ca2+]i in the olfactory receptor cells in nominally Ca2+ free solution and stimulated by amyl acetate. When 3 mM CaCl2 was added, the [Ca2+]i increased remarkably. When cells were stimulated with amyl acetate in a CaCl2-depleted extracellular fluid supplemented with 10 microM verapamil (an L-type voltage-dependent calcium channel antagonist), no change occurred in [Ca2+]i. The addition of CaCl2 to this fluid did not elevate [Ca2+]i, in any cell. This would suggest that the [Ca2+]i increase in the presence of amyl acetate may mainly depend on the influx of calcium from the extracellular space. Our finding that the influx of extracellular calcium into cells was suppressed by verapamil suggests that the calcium channel, which is opened by amyl acetate, is a voltage-dependent L-type Ca2+ channel.

Animals↗
PubMed1979

Microcinematographic demonstration of synchronous and asynchronous myoepithelial contractions in mouse submandibular gland rudiments in organotypic culture.

Time-lapse phase-contrast cinematography revealed contractile activity within mouse submandibular salivary gland rudiments in organotypic culture. Three types of contraction were distinguishable. In type I (voiding contractions), all portions of the gland contracted synchronously, and the active state ranged from 30 min to 2 hr. In type II (priming contractions), all portions of the gland contracted synchronously, but the active state was shorter, ranging from 4 to 10 min. In type III (churning contractions), isolated foci in lobules or secretory units throughout the gland contracted asynchronously and had very short active states of about 1 min. By electron microscopy, myoepithelial cells could first be demonstrated in submandibular glands developing either in vitro or in vivo, at 21 days postconception. Contractions in the cultured rudiments began as early as 18 days postconception. Since neither smooth nor striated muscle could be identified in these glands by electron microscopy, the contractions are believed to result from myoepithelial activity that apparently may begin before ultrastructural evidence of myoepithelial differentiation is clearly present. Although, for over a century, myoepithelium has been presumed to have a contractile function and indirect evidence has lent ample support to this presumption, the present study represents the first direct cinematographic demonstration and characterization of myoepithelial contractions, under conditions in vitro.

Animals↗
PubMed1978

Monolayer cultures of rat pituitary intermediate lobe: growth and fine structure of epithelial cells.

Rat hypophyseal intermediate lobe cells, partially dispersed by trypsin, were cultivated on an inert surface. The attaching capacity, the growth of the cultures and the fine structure of the epithelial cells developed during cultivation were studied. The majority of the cell groups attached to the inert surface within 72 h after explantation. Epithelial islands developed, with a layer consisting of fibroblast-like cells beginning to form at their periphery. By the end of the second week of cultivation the epithelial islets and fibroblast-like cells formed a monolayer. The epithelial cells survived for five weeks. The granular cytoplasm of the epithelial island cells could be well identified on the phase-contrast microcinematographic frames of the young cultures; both epithelial and fibroblast-like cells divided. The mitotic ability of both cell types could be confirmed by their tritiated-thymidine incorporation, too. In young cultures, the fine-structural features of epithelial cells were practically identical with those of the glandular cells in intact glands. In older cultures, the secretory granules in the epithelial cells were lower in number; the localization of the granules at the periphery was predominating.

Animals↗