Cytology of Spore Germination in Clostridium pectinovorum

The process of spore germination in Clostridium pectinovorum has been followed by phase-contrast and electron microscopy. Unlike most other Bacillaceae, germination of this species takes place within the sporangium. Under phase-contrast, the spore darkens and swells slightly, and then the vegetative rod slips out through the end opposite the collar-like extension of the sporangium. In thin sections, a spore from an early stage in germination consists of a central protoplast, core membrane, germ cell wall, cortex, and two coats. Within a short period, the cortex disintegrates and the young cell develops. It possesses a large fibrillar nucleoplasm and several mesosomes. Subsequently, the young cell elongates, becomes somewhat deformed, and then emerges through a narrow aperture in the inflexible coats of the spore, finally rupturing the sporangium. Free vegetative cells of C. pectinovorum resemble in their structure other gram-positive rods.     

Intermediate (10 nm) filaments in human malignant mesothelioma.

Human malignant mesotheliomas were studied by electron microscopy. Three main types of cells were seen--submesothelial epithelioid cells, epithelial lining cells and fibroblast-like cells. In submesothelial epithelioid cells prominent arrays of intermediate (10 nm) filaments were often seen attached to plasma membrane, mitochondria, nuclei and concentric whorls of rough endoplasmic reticulum. The other types of cell found in the tumors, epithelial lining cells and fibroblast-like cells, lacked such distinct filaments. The intermediate filaments were especially abundant in cells with extensive whorling of endoplasmic reticulum. The association of intermediate filaments with such deranged cytoplasmic organization suggests that they play a role in the altered behavior of malignant cells.     

Bacterial plasmolysis as a physical indicator of viability.

Bacterial plasmolytic response to osmotic stress was evaluated as a physical indicator of membrane integrity and hence cellular viability. Digital image analysis and either low-magnification dark-field, high-magnification phase-contrast, or confocal laser microscopy, in conjunction with pulse application of a 1.5 M NaCl solution, were used as a rapid, growth-independent method for quantifying the viability of attached biofilm bacteria. Bacteria were considered viable if they were capable of plasmolysis, as quantified by changes in cell area or light scattering. When viable Salmonella enteritidis biofilm cells were exposed to 1.5 M NaCl, an approximately 50% reduction in cell protoplast area (as determined by high-magnification phase-contrast microscopy) was observed. In contrast, heat- and formalin-killed S. enteritidis cells were unresponsive to NaCl treatment. Furthermore, the mean dark-field cell area of a viable, sessile population of Pseudomonas fluorescens cells (approximately 1,100 cells) increased by 50% as a result of salt stress, from 1,035 +/- 162 to 1,588 +/- 284 microns2, because of increased light scattering of the condensed, plasmolyzed cell protoplast. Light scattering of ethanol-killed control biofilm cells underwent little change following salt stress. When the results obtained with scanning confocal laser microscopy and a fluorescent viability probe were compared with the accuracy of plasmolysis as a viability indicator, it was found that the two methods were in close agreement. Used alone or in conjunction with fluorochemical probes, physical indicators of membrane integrity provided a rapid, direct, growth-independent method for determining the viability of biofilm bacteria known to undergo plasmolysis, and this method may have value during efficacy testing of biocides and other antimicrobial agents when nondestructive time course analyses are required.     

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.     

Ultrastructure of Mycoplasma laidlawii During Culture Development

Mycoplasma laidlawii B has been studied by phase-contrast and electron microscopy (thin-section and negative staining). Exponential-phase cells are filaments with the following structures: surface unit membrane, nuclear material, ribosomes, and intracellular granular region. These cells appear to reproduce by binary fission. In stationary phase, the filamentous cells have numerous constrictions, giving them a beaded appearance. Nonviable death-phase cultures contain cellular debris: swollen cells, granular bodies, and other aberrant forms.     

Use of immunofluorescence and phase-contrast microscopy for detection and identification of Giardia cysts in water samples

A method was developed in which indirect immunofluorescence and phase-contrast microscopy are used for rapid detection and identification of Giardia cysts in raw and finished water supplies. When anti-Giardia cyst antiserum and fluorescein conjugate were applied to known Giardia cysts on membrane filters, the cysts fluoresced bright green when they were illuminated by UV light. This procedure permitted individual cysts to be quickly located even in samples heavily contaminated with other microorganisms and debris. The identity of presumptive Giardia cysts located in this way could then be confirmed by observing characteristic internal morphological features with phase-contrast microscopy. With this method, Giardia cysts were detected and their identities were confirmed in samples taken from raw and finished surface water supplies during several recent outbreaks.     

Use of immunofluorescence and phase-contrast microscopy for detection and identification of Giardia cysts in water samples.

A method was developed in which indirect immunofluorescence and phase-contrast microscopy are used for rapid detection and identification of Giardia cysts in raw and finished water supplies. When anti-Giardia cyst antiserum and fluorescein conjugate were applied to known Giardia cysts on membrane filters, the cysts fluoresced bright green when they were illuminated by UV light. This procedure permitted individual cysts to be quickly located even in samples heavily contaminated with other microorganisms and debris. The identity of presumptive Giardia cysts located in this way could then be confirmed by observing characteristic internal morphological features with phase-contrast microscopy. With this method, Giardia cysts were detected and their identities were confirmed in samples taken from raw and finished surface water supplies during several recent outbreaks.     

Characterization of human mammary cell types in primary culture: Immunofluorescent and immunocytochemical indicators of cellular heterogeneity

Summary Parenchymal organoidal structures that were obtained from collagenase digestion of reduction mammoplasty specimens of apparently normal human breasts have been grown in short-term primary cultures, either on plastic or on floating gels of polymerized rat-tail collagen. Three morphologically distinct major cell types are readily observed in both systems: cuboidal cells, which occupy apical positions on collagen gels; larger, epithelioid, or basal cells on gels; and elongated cells which penetrate into the gel. In addition, a fourth cell type, that of a large, flat cell, is observed less readily by phase contrast microscopy on the surface of cultures grown on plastic. Immunofluorescent and immunocytochemical staining of cultures on plastic or histologic sections of cultures on gels have been undertaken with antisera and other histochemical reagents that stain the different parenchymal cell types in vivo. Thus antisera to epithelial membrane antigen(s), monoclonal antibodies (MABs) to the defatted mammary milk fat globule membrane, peanut lectin, and keratin MAB LE61, which preferentially stain the epithelial cells of ducts in vivo, also stain the cuboidal/apical cells in vitro. The large, flat cells are stained intensely by the first three reagents but not by the last one. Antisera to collagen IV, laminin, fibronectin, actin, keratin MAB LP34, MABs to the common acute lymphoblastic leukemia antigen, and MAB LICR-LON-23.10, which showed enhanced staining for the ductal myoepithelial cells in vivo, also stain the epithelioid/elongated cells in vitro. However, the effect of the last four reagents is reduced considerably in most elongated cells, and MAB LP34 stains the large, flat cells intensely. Heterogeneous cells of intermediate morphologies and staining patterns between the cuboidal/flat cells and large epithelioid cells have also been identified. The results suggest that the cuboidal cells and large, flat cells are related to mammary epithelial cells, whereas the large epithelioid/elongated cells have some characteristics of myoepithelial cells, and that intermediate forms may exist in culture between the two parenchymal cell types. This work was supported in part by the Ludwig Institute for Cancer Research and the Cancer and Polio Research Fund. Dr. M. J. Warburton is supported by the Cancer Research Campaign.     

Immunofluorescence and inhibitor studies on creatine kinase and mitosis

Immunofluorescence microscopy of mitotiv PtKl cells with antibody against M-line creatine kinase from chicken breast muscle revealed spindle staining largely identical with that of antibody against tubulin. Treatment with epoxycreatine, a creatine analog and covalent creatine kinase inhibitor, allowed the study of creatine kinase function in these non-muscle cells. Cells fixed at interphase following epoxycreatine treatment maintained actin stress fibers and creatine kinase localization on intermediate filaments. Treated prometaphase cells lacked spindles as observed by immunofluorescence microscopy, but displayed instead a central region of diffuse immunofluorescent staining surrounded by condensed chromosomes. Those observed in metaphase or anaphase appeared normal by phase-contrast microscopy, but lacked immunofluorescent staining. Living epoxycreatine-treated cells were observed by phase-contrast microscopy to form spindles and progress through mitosis following an extended prometaphase.     

Destruction of the outer membrane permeability barrier of Escherichia coli by heat treatment

Heat treatment of a wild-type Escherichia coli strain at 55 degrees C in 50 mM Tris-hydrochloride buffer with or without 10 mM magnesium sulfate or HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer at pH 8.0 caused an increase in cell surface hydrophobicity. By determining the location of n-hexadecane droplets attached to cells by phase-contrast microscopy, the septal and polar regions of heated cells appeared to become the most frequently hydrophobic. Some of the lipopolysaccharide molecules in the outer membrane were released from heated cells, and the cells became susceptible to the hydrolytic action of added phospholipase C. Heat-treated cells also became permeable to the hydrophobic dye crystal violet, which was added externally. The release of part of the outer membrane by heat treatment appeared to bring about the disorganization of the outer membrane structure and, as a consequence, to result in the partial disruption of the permeability barrier function of the outer membrane. Tris was found to enhance damage to the outer membrane by heat.     

Destruction of the outer membrane permeability barrier of Escherichia coli by heat treatment.

Heat treatment of a wild-type Escherichia coli strain at 55 degrees C in 50 mM Tris-hydrochloride buffer with or without 10 mM magnesium sulfate or HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer at pH 8.0 caused an increase in cell surface hydrophobicity. By determining the location of n-hexadecane droplets attached to cells by phase-contrast microscopy, the septal and polar regions of heated cells appeared to become the most frequently hydrophobic. Some of the lipopolysaccharide molecules in the outer membrane were released from heated cells, and the cells became susceptible to the hydrolytic action of added phospholipase C. Heat-treated cells also became permeable to the hydrophobic dye crystal violet, which was added externally. The release of part of the outer membrane by heat treatment appeared to bring about the disorganization of the outer membrane structure and, as a consequence, to result in the partial disruption of the permeability barrier function of the outer membrane. Tris was found to enhance damage to the outer membrane by heat.     

Cell division and trichome breakage in Beggiatoa

The process ofBeggiatoa trichome division was elucidated through phase-contrast microscopy and transmission and scanning electron microscopy. Trichome breakage and dispersion is accomplished by the formation of sacrificial cells (necridia) at various points within the trichome. Upon dying, the sacrificial cells lyse, dividing the trichome into two daughter trichomes. This process is identical with that found in many oscillatorian blue-green bacteria, but differs from the mechanism of trichome division in most of the other flexuous gliding bacteria. Cellular division within the trichome occurs by septation, involving the cytoplasmic membrane and the electron-dense L2 (peptidoglycan) layer. The outer envelope layers do not take part in division.     

Smashed fission yeast walls

Twenty-three samples of fission yeast cells (Schizosaccharomyces pombe) were smashed by shaking them with glass beads. The samples represented all phases of the culture cycle, with the lag and log phases emphasized. Ruptured walls of the smashed cells were observed by phase-contrast and electron microscopy. Ruptures were tabulated with respect to their magnitudes and locations. Ruptures occurred not at random, nor at sites directed by geometry, but predominated in certain definable wall regions. These discontinuities were correlated with morphogenetic activities of the cell. Thus, the extensile end was found to be most fragile through most of the culture cycle. Also fragile was the nonextensile end, its edge more than its middle. Further, the data were applied to the testing of predictions from extant models (Johnson endohydrolytic softening model and Wessels presoftened-posthardened and crosslinking model) for hyphal tip extension. The frequency of rupture at the extensile (old) end of the cell was qualitatively predicted by both models; the frequency at the nonextensile (new) end was not predictable by either. Rupture frequencies and characteristics at other regions conformed to predictions by one or the other model, but rarely by both.     

Direct Epoxy Embedding for Vertical Sectioning of Cells Grown as a Monolayer on Millipore Filter

Cells cultured as a monolayer on MF-Millpore GSWP. 0.22 μ pore size, filter were fixed, dehydrated, and examined by phase-contrast microscopy with the filter immersed in a 1:1 mixture of xylene and the embedding medium. The membrane was cut into 2 × 20 mm strips, and each strip which was selected for desired cells was embedded vertically in a BEEM capsule. Thus direct embedding which allowed edgewise sectioning of cells was obtained without removing them from the culturing support.     

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.     

Klebsiella pneumoniae adhesion to epithelioid cells in vitro

The adhesion of K. pneumoniae K24 capsular strain No. 6723 onto subcultured epithelioid human kidney cells RN was studied overtime by light microscopy and by transmission and scanning electron microscopy. To find out the bacterial capsule and glycocalyx of epithelioid cells, the method of staining the samples with ruthenium red was used, this stain producing the coloration of extracellular acidic mucopolusaccharides . The bacteria were found to attach to the qlycocalyx of epithelioid cells by means of protruding areas on the capsule which retained its form and size after both stabilization with ruthenium red and standard glutar -osmium fixation. Under the action of the bacteria epithelioid cells were found to round off, become longer and increase the number of processes. At the sites of contact with the bacteria specific short cytoplasmic processes serving for the attachment of K. pneumoniae cells were discovered.     

Effect of chromosomal breaks induced by x-irradiation on the number of mesosomes and the cytoplasmic organization of Streptococcus faecalis

A model which explains mesosome formation via a contraction of the cytoplasm and nucleoid when bacteria are physiologically disturbed was tested by: (1) X-irradiation of unfixed cells of Streptococcus faecalis to produce chromosomal breaks and to remove DNA attached to the cell membrane; (2) subsequent determination of the number of irradiated cells in which mesosomes (using electron microscopy) and central density changes (using phase-contrast microscopy) could be visualized after fixative was added. Results obtained by exposure of cells to doses up to 1100 krads before fixation indicated that: (1) the number of cells with central mesosomes was reduced proportional to the decrease in the molecular weight of the DNA due to double-strand breaks: (2) the number of cells with total (central plus peripheral) mesosomes and the number of cells with peripheral mesosomes were both reduced proportional to the removal of DNA attached to the cell membrane (M band); (3) the nucleoid became more diffusely organized. Exposure of cells to doses greater than 1100 krads before fixation resulted in: (1) an increase in the number of cells with central and peripheral mesosomes (compared to cells exposed to lower dosages); (2) a return to the centralized, dense nucleoid seen in unirradiated cells.These results suggest that mesosomes are formed when localized sites on the cell membrane are pulled from close contact with the cell wall into the cytoplasm by the action of a cross-linking fixative via the aggregation of intracytoplasmic components such as DNA. This model considers the attachment of DNA and/or other cytoplasmic components to the membrane as an intrinsic part of its mechanism. The formation of central and peripheral mesosomes in unirradiated and X-irradiated cells are contrasted.     

Ultrastructural and morphometric aspects of the juxtaglomerular apparatus in the monkey kidney

Summary The authors describe the ultrastructure of the juxtaglomerular apparatus in five adult male Cebus apella monkeys and communicate morphometric data of the macula densa.In comparison with several species of rodents examined before, the macula cells of the monkey contain many more mitochondria and possess a particularly thick basal membrane. The relative volume of the nuclei is slightly smaller than in rodents.The Goormaghtigh cells of the monkey resemble those of the other animals investigated.The epithelioid (or juxtaglomerular) cells do not contain secretory granules. This observation reminds one of the behavior of the epithelioid cells of guinea pigs.Dedicated to Professor Dr. med. G. Petry (Marburg/Lahn) on the occasion of his 65th birthdayThe authors wish to thank Professor Dr. G. Peters, Head of the Institute of Pharmacology, and Professor Dr. F. Roch-Ramel for kindly having provided the monkey kidneys     

Unusual ultrastructural features in three strains of Cyanothece (cyanobacteria)

Three unicellular cyanobacterial strains (PCC 7425, PCC 8303, PCC 9308) assigned to the genus Cyanothece Komárek 1976, which showed an unusually high content of light refractile inclusions when viewed by phase-contrast microscopy, were characterized by confocal laser scanning microscopy and transmission electron microscopy. All strains had concentric cortical thylakoids and a compact central nucleoid. Frequently, the two innermost thylakoid membranes protruded to form circular enclosures containing cytoplasm or electron-transparent granules, or both. The largest granules were partially immersed in the nucleoid region, but they remained attached to the inner cortical thylakoids by a single narrow connection. The pattern of binary cell division in strain PCC 7425 was different than that in strains PCC 8303 and PCC 9308. In the former, all cell wall layers invaginated simultaneously, whereas in the latter the invagination of the outer membrane was delayed compared to that of the cytoplasmic membrane and the peptidoglycan layer. Thus, prior to completion of cell division, the new daughter cells of strains PCC 8303 and PCC 9308 were transiently connected by a thick septum, which was not observed in strain PCC 7425. Nucleoid partitioning coincided with initiation of cell division in all three strains and was unlike that reported in other bacteria and in archaea, in which separation of the nucleoids precedes cell division. Based on the common morphological and ultrastructural features, the three strains of Cyanothece examined constitute a distinct cluster, which might deserve independent generic status.     

Fetal mouse lung in circumfusion system cultures.

Fetal mouse lungs were cultivated, using the dual-rotary circumfusion system for tissue culture, and their histotypic development was surveyed for 75 days by phase-contrast and electron microscopy. Alveoli, terminal bronchioles and alveolar macrophages were photographed periodically with still and time-lapse phase-contrast microscopy. Their histotypic appearance was confirmed by electron micrographs of the 1- and 2 1/2-month-old specimens. These revealed typical alveoli surrounded by a basal lamina and composed of types I and II pneumocytes containing various lamellar-body forms within the type II cells, the alveolar lumen, and the alveolar macrophages. There was a shift from almost all type II cells in the 1-month-old alveoli to the presence of frequent type I cells as constituents of the alveoli in the 2 1/2-month-old cultures. The terminal bronchioles were tubules consisting of ciliated cells with Clara cells interspersed between them. The ciliated cells contained as many as 30 cilia or basal bodies per section and numerous microvilli. They were attached to each other and to the Clara cells by junctional complexes and accessory desmosomes which were generally in the apical ends of the cells. The Clara cells typically had glycogen granules interspersed between lamellae of the endoplasmic reticulum, contained numerous well dispersed mitochondria, occasional lysosome-like granules and crystalloid bodies which appeared to be tubular. Some Clara cells presented a moderatley dense secretory granule in the center of the whorl of the endoplasmic reticulum.