Ultrastructure of Cells Cultured on Polycarbonate Membranes

A method is described for preparing undisturbed cell cultures for both scanning and transmission electron microscopy. Cells were propagated on polycarbonate membranes with pores of 0.2 pm or less. Cultured cells together with their supports were prepared for both scanning electron microscopy and transmission electron microscopy using routine methods. For transmission electron microscopy a rapid schedule of infiltration and polymerization was used. The method described in this report yielded good results and it allowed the fine structure of cultured cells to be viewed in situ by both scanning electron microscopy and transmission electron microscopy.     

Conditions critical for optimal visualization of bacteriophage adsorbed to bacterial surfaces by scanning electron microscopy.

The potential of scanning electron microscopy as a tool for the detection of viruses on cell surfaces has been studied using bacteriophage P1 adsorbed to Shigella dysenteriae as a model system. Viral particles were readily detectable by scanning electron microscopy on the surface of infected cells which were fixed with glutaraldehyde followed by postfixation in OsO4 and prepared by critical point drying. The virus-studded surface of the infected cells differed markedly from the relatively smooth surfaces of uninfected control cells. Examination of the same preparations with transmission electron microscopy revealed numerous viral particles adsorbed to the surfaces of infected cells, whereas the control cells were free of viruses as expected. Glutaraldehyde fixation alone did not preserve the surface detail of infected cells: cells adsorbed with viruses were not distinguishable from control cells by scanning electron microscopy although by transmission electron microscopy viruses could be visualized. Air drying from water or absolute alcohol resulted in unsatisfactory preservation as compared to the appearance of infected cells prepared by the critical point method. Thus, scanning electron microscopy is capable of resolving viral particles on cell surfaces, but detection of these particles is completely dependent both on the method of fixation and on the technique of drying used.     

A Method for the Examination of the Same Cell Using Light, Scanning and Transmission Electron Microscopy

A method is described for preparing the same cell from a cytospin preparation for comparative investigation by light microscopy, scanning electron microscopy and transmission electron microscopy. A permanent numbered grid pattern was etched on a glass microscope slide to facilitate cell location in each microscopic mode. Data from one cell or group of cells was thus obtained from three sources. This method provides a useful adjunct to routine cytological diagnosis.     

Use of a Technovit 7200 VLC to Facilitate Integrated Determination of Aluminum by Light and Electron Microscopy

Technovit 7200 VLC is an excellent embedding medium for both inorganic histochemistry by light microscopy and X-ray microanalysis by scanning and transmission electron microscopy. Liver samples from rats after intraperitoneal treatment with aluminum chloride were fixed in glutaraldehyde and embedded in the resin. Thick sections were easily cut on an ultramicrotome and stained with aluminon for aluminum (Al). An intense positive reaction with aluminon was observed in the Kupffer cells by light microscopy. The surface structures of the same resin block cut for light microscopy were observed under a scanning electron microscope fitted with an energy dispersive X-ray spectrometer. The Kupffer cells appeared white in the backscattered mode. Localization of Al in the Kupffer cells was confirmed by an X-ray distribution map in the scanning electron microscope. Subcellular localization of Al in the Kupffer cells was performed on the same semithin sections using a transmission electron microscope equipped with an energy dispersive X-ray spectrometer. Most Al was found in lysosomes of the Kupffer cells. The resin was stable in the electron beam and chlorine-free.     

Ultrastructural changes in murine lymphocytes induced by aflatoxin B1

This investigation sought to determine whether splenic lymphocytes obtained from Balb/C mice exposed to aflatoxin B1 (AFB1) showed any ultrastructural changes which could account for the immunodysfunction attributable to aflatoxins. Lymphocytes obtained from Balb/C mice administered aflatoxin B1 in olive oil daily for three weeks were studied using both transmission and scanning electron microscopy. The lymphocytes demonstrated ultrastructural changes primarily in the mitochondria where marked internal dissociation of the cristae was revealed by transmission electron microscopy. All other cellular organelles were unaffected. No significant alterations in external structure were observed under scanning electron microscopy. The findings of this study indicate that AFB1 administration does not affect the surface topography of lymphocytes, but AFB1, by causing extensive mitochondrial damage, may affect the way in which these cells function. This could be a possible explanation for the immunodysfunction associated with AFB1.Abbreviations AFB1 Aflatoxin B1 - SEM scanning electron microscopy - TEM transmission electron microscopy     

Electron probe X-ray microanalysis of cultured myogenic C2C12 cells with scanning and scanning transmission electron microscopy

Heterogeneity of the elemental content of myogenic C2C12 cultured cells was studied by electron probe X-ray microanalysis (EPXMA) with scanning (SEM EPXMA) and scanning transmission electron microscopy (STEM EPXMA). The best plastic substrate for growing cells was Thermanox. For STEM EPXMA, a Formvar film coated with carbon was found to be suitable substrate. The cells examined by scanning transmission electron microscopy showed great heterogeneity in their elemental content in comparison with the cells examined in the scanning electron microscope despite of an almost identical preparation procedure for EPXMA. Nevertheless the K/Na ratios obtained from both methods of EPXMA were very close (4.1 and 4.3). We conclude that the observed discrepancy in the elemental content obtained by the two methods may be due to differences in instrumentation and this must be taken into account when planning a comparative study.     

Colloidal gold, a useful marker for transmission and scanning electron microscopy.

Electron dense markers of a size suitable for transmission electron microscopy and scanning electron microscopy have been prepared with gold granules labeled with a monolayer of specific macromolecules. The optimum conditions for preparing the markers have been ascertained. The method is simple, rapid and seems to be general since gold granules have been labeled with polysaccharides and proteins. As homogeneous populations of gold granules having different sizes can be prepared, the method is also suitable for double marking experiments. The gold technique is illustrated by the localization of polysaccharides and glycoproteins on yeast cell walls and erythrocyte membranes by transmission electron microscopy and on yeast cells and intact erythrocytes by scanning electron microscopy. Good spatial resolution of the marker was achieved in all cases. The method is also suitable for marking thin sections. Spectrophotometric measurements were used to determine the number of gold granules adsorbed per cell.     

Use of electron microscopy to characterize the surfaces of flocculent and nonflocculent yeast cells

The surfaces of flocculent and nonflocculent yeast cells have been examined by electron microscopy. Nonextractive preparative procedures for scanning electron microscopy allow comparison in which sharp or softened images of surface details (scars, etc.) are the criteria for relative abundance of flocculum material. Asexually flocculent budding-yeast cells cannot be distinguished from nonflocculent budding-yeast cells in scanning electron micrographs because the scar details of both are well resolved, being hard and sharp. On the other hand, flocculent fission-yeast cells are readily distinguished from nonflocculent cells because fission scars are mostly soft or obscured on flocculent cells, but sharp on nonflocculent cells. Sexually and asexually flocculent fission-yeast cells cannot be distinguished from one another as both are heavily clad in "mucilaginous" or "hairy" coverings. Examination of lightly extracted and heavily extracted flocculent fission-yeast cells by transmission electron microscopy provides micrographs consistent with the scanning electron micrographs.     

Comparison of tissue-cultured bovine endothelial cells from aorta and sphenous vein

Summary Endothelial cells were harvested from bovine aorta and saphenous vein with collagenase and cultured in McCoy's 5a medium (modified GIBCO) supplemented with 10% fetal bovine serum. The cells were subcultured through 17 passages over 4 to 5 months. The growth properties in culture of the two cell types were compared. Morphological comparisons included phase microscopy and scanning and transmission electron microscopy. Comparisons with cultured aortic smooth-muscle cells were made using phase and scanning electron microscopy. No differences were found between cultured endothelial cells from aorta and saphenous vein. Differences in growth patterns in culture clearly distinguished both endothelial cell types from smooth-muscle cells. The presence of Weibel-Palade bodies identified the cells from both sources as endothelial. This work was supproted by Grants HL-1330 and HL-17269 from NIH.     

A fast screening method for surface layers on Gram-positive bacteria

A two-step screening method is described to identify regularly arranged surface layers (S layers) on Gram-positive bacterial strains. A non-destructive release of S-layer sheets is achieved by enzymatic hydrolysis of the underlying peptidoglycan using lysozyme. The existence of regular S layers is then directly confirmed by scanning force microscopy or transmission electron microscopy. This method requires a minimal amount of bacterial cells and may be used as a `quick test' for demonstrating the presence of S layers.     

Fixation of platelets for scanning and transmission electron microscopy.

A double fixation method of preparing platelet suspensions for both scanning and transmission electron microscopy is outlined. Prefixation in 0.1% glutaraldehyde allows for immediate preservation of morphologic characteristics induced by experimental procedures, but does not completely destroy platelet surface stickiness. Preservation of surface stickiness allows subsequent production of a platelet pellet for processing for transmission electron microscopy. This pelleting cannot be achieved when higher initial concentrations of glutaraldehyde are used for prefixation. Prefixation in 0.1% glutaraldehyde is also an appropriate initial step for preservation of platelets in suspension for scanning electron microscopy.     

Fixation of Platelets for Scanning and Transmission Electron Microscopy

A double fixation method of preparing platelet suspensions for both scanning and transmission electron microscopy is outlined. Prefixation in 0.1% glutaraldehyde allows for immediate preservation of morphologic characteristics induced by experimental procedures, but does not completely destroy platelet surface stickiness. Preservation of surface stickiness allows subsequent production of a platelet pellet for processing for transmission electron microscopy. This pelleting cannot be achieved when higher initial concentrations of glutaraldehyde are used for prefixation. Prefixation in 0.1% glutaraldehyde is also an appropriate initial step for preservation of platelets in suspension for scanning electron microscopy.     

In vivo adherence of Pseudomonas aeruginosa to rat bladder epithelium

To date, the study of bacterial adherence to uroepithelial cells has utilized considerable variation in methodology, resulting in highly divergent conclusions. In an attempt to standardize methodology, a modified method is described to more accurately measure the in vivo bacterial adherence to rat bladder uroepithelium utilizing Pseudomonas species labeled with 2-[3H]adenine. Two isolates of P. aeruginosa were selected for more intensive study; one showed consistently good adherence; the second strain always adhered poorly, thus providing a negative control. Maximum adherence was detected after 60 min of incubation. Neither of the two Pseudomonas isolates when examined by transmission electron microscopy showed evidence of pili formation. The morphological features of Pseudomonas adherence to bladder mucosa as studied by scanning electron microscopy are described.     

Positive detection of mycoplasma contamination by the whole-mount preparation of cell cultures for transmission electron microscopy.

Low-level mycoplasma contamination of cell cultures is difficult to recognize with presently available techniques. This report describes the adaptation of the whole-mount technique, usually used for scanning microscopy, for transmission electron microscopy. The differentiation between microvilli and the equal-sized filamentous mycoplasma is based on the differential density obtained by the use of the method described. This method allows positive identification of mycoplasma and reduces the preparation time and the time necessary for scanning the preparation.     

Fine structure of the dorsal lingual epithelium of the frog, Rana rugosa

Scanning and transmission electron microscopy was employed to investigate the ultrastructure of the lingual dorsal epithelial cells of the frog, Rana rugosa. The specimens for scanning electron microscopy were prepared by a method that involved osmium postfixation and treatment with acid to remove extracellular material that adhered to the surface of the tongue. Over almost the entire dorsal surface, filiform papillae, consisting of a large number of non-ciliated cells with microridges and a very small number of ciliated cells, were compactly distributed. Fungiform papillae were scattered among these filiform papillae. A round sensory disk was located on the top of each fungiform papilla. Each sensory disk was encircled by a band of ciliated cells. Transmission electron microscopy revealed that a large part of the filiform papillar epithelium was composed of cells that contained numerous electron-dense granules. These cells were coincident with the non-ciliated cells observed by scanning electron microscopy. In these cells, the nucleus was located on the basal side, and the ergastoplasm was well-developed on the basal side of the nucleus.     

Comparison of tissue-cultured bovine endothelial cells from aorta and saphenous vein

Endothelial cells were harvested from bovine aorta and saphenous vein with collagenase and cultured in McCoy's 5a medium (modified GIBCO) supplemented with 10% fetal bovine serum. The cells were subcultured through 17 passages over 4 to 5 months. The growth properties in culture of the two cell types were compared. Morphological comparisons included phase microscopy and scanning and transmission electron microscopy. Comparisons with cultured aortic smooth-muscle cells were made using phase and scanning electron microscopy. No differences were found between cultured endothelial cells from aorta and saphenous vein. Differences in growth patterns in culture clearly distinguished both endothelial cell types from smooth-muscle cells. The presence of Weibel-Palade bodies identified the cells from both sources as endothelial.     

Three methods for isolating viable anthozoan endoderm cells with their intracellular symbiotic dinoflagellates

Three maceration methods are described for the isolation of single endoderm cells from marine cnidarians. Two are enzymatic treatments suitable for fleshy anthozoans such as sea anemones and zoanthids. The third employs calcium free sea water and is suitable for stony corals. The viability and morphology of the endoderm cells is described using fluorogenic dyes and scanning and transmission electron microscopy.     

Initial attachment of Candida albicans cells to buccal epithelial cells. Demonstration of ultrastructure with the rapid-freezing technique

The rapid-freezing technique was applied in association with scanning and transmission electron microscopy to observe the initial attachment (or contact) of Candida albicans cells to exfoliated human buccal epithelial cells. Low temperature scanning electron microscopy provided detailed three-dimensional morphological features of the yeast-epithelial cell association; adhesion of C. albicans cells to host cells was primarily owing to an interaction between fibrillar layer of the yeast cell wall and the membrane interdigitations of the epithelial cells. Such a particular interconnection between the two cells was confirmed by the freeze-substitution fixation for transmission electron microscopy. These results clearly demonstrate the outermost fibrillar cell wall layer of C. albicans responsible for adhesion to host cells.     

The leucocytes of the elasmobranch Scyliorhinus canicula L.—a morphological study

The leucocytes of the peripheral blood of the elasmobranch Scyliorhinus canicula L. were examined by light, transmission and scanning electron microscopy and histochemistry. Seven distinct leucocytes were identified including lymphocytes, thrombocytes, monocytes and granulocytes. The major characteristics of these cells and their relative distribution is described.     

Enrichment of perforate septal pore caps from the basidiomycetous fungus Rhizoctonia solani by combined use of French press, isopycnic centrifugation, and Triton X-100

Septal pore caps occur in many filamentous basidiomycetes located at both sides of the dolipore septum and are at their base connected to the endoplasmic reticulum. The septal pore cap ultrastructure has been described extensively by the use of electron microscopy, but its composition and function are not yet known. To enable biochemical and functional analyses in the future, we here describe an enrichment method for perforate septal pore caps from Rhizoctonia solani. Our method is based on the combined use of French press and isopycnic centrifugation, using a discontinuous sucrose gradient followed by a treatment with Triton X-100. Enrichment was monitored by the use of scanning electron microscopy and transmission electron microscopy. Using the same isolation method, smaller septal pore caps were isolated from two other basidiomycetes as well. Furthermore, we showed pore-occluding material co-purified with the septal pore caps. This observation supports the hypothesis that septal pore caps play a key role in the plugging process of the septal pores in filamentous basidiomycetes.