Colicin Typing of Shigella sonnei

The relative amount of extracellular polymer which remains about Azotobacter vinelandii, Zoogloea ramigera, Klebsiella pneumoniae, and Diplococcus pneumoniae after critical-point drying was studied by electron microscopy. The results obtained with this technique are compared to those obtained with methods that illustrate extracellular polymer, such as freeze-etching and ruthenium red staining. Comparative results indicate critical-point drying to be a rapid, reliable method for the determination of capsule-like polymer surrounding bacterial cells. In addition, critical-point drying can be used to observe morphogenetic changes, such as vesicle production.     

Fine structure and distribution of extracellular polymer surrounding selected aerobic bacteria.

The structure and distribution of extracellular polymer surrounding Bacillus circulans, Diplococcus (Streptococcus) pneumoniae, Streptococcus salivarius, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Herella vaginacola (Acinetobacter calcoaceticus), and Agrobacterium tumefaciens were studied by electron microscopy. A modified ruthenium red staining procedure was used to examine the fine structure of capsule and slime. Freeze-etching and critical-point drying were used to examine the quantity of unaltered exocellular material. Comparative data demonstrate that fibrillar extracellular polymer surrounding B. circulans, D. pneumoniae, and K. pneumoniae is capsule (cell wall attached) which is characteristic of the producing organism. Capsular polymer generally appeared fibrillar, although globular polymer consisted of capsular subunits bound to S. salivarius and H. vaginacola. Exocellular slime was present about S. aureus, P. aeruginosa, and A. tumefaciens.     

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.     

Cell-to-cell tracer movement in cardiac muscle

Summary An attempt was made to label injured cardiac muscle cells by exposing them to two electron-opaque tracers, ruthenium red and lanthanum nitrate. To do this, false tendons of sheep hearts containing strands of Purkinje fibers were sectioned, allowed to heal, and then exposed to the tracer during fixation. After this treatment, a group of cells near the cut end were found to be labelled intracellularly with the tracers while the remaining cells in the strand were unlabelled.For comparison, several false tendons were fixed briefly in glutaraldehyde before being cut and then exposed to the tracer. With lanthanum, the results were similar to those obtained when the cells had been damaged prior to fixation. However, when ruthenium red was used as the tracer, it penetrated much further into the cellular strand, its intensity gradually diminishing with distance from the cut end. This finding of apparent dye-coupling in fixed tissue was surprising since it has been suggested that glutaraldehyde fixation converts all communicating junctions to the uncoupled state.Dye-coupling of fixed tissue with ruthenium red as a tracer was seen also in frog atrial trabeculae.Gap junctions between injured (and presumably uncoupled) sheep heart Purkinje cells were compared to gap junctions between uninjured control cells in thin sections. No difference was detected.     

Ruthenium Red—p-Phenylenediamine Staining of Monolayers to Facilitate Handling and Selection of Specific Cells for Transmission Electron Microscopy

The handling of monolayers for transmission electron microscopy has presented many problems, the main one being difficulty in visualizing the monolayers after polymerization of their plastic embedment following conventional glutaraldehyde-osmium fixation.

The application of ruthenium red—p-phenylenediamine during processing produced intensely darkened cells which could be examined and photographed either in 95% ethanol or following Spurr embedment without further treatment or sectioning. This treatment also facilitated orientation of the monolayers when re-embedding, and permitted precise localization of monolayers within flat embedding molds when trimming and thin sectioning for transmission electron microscopy.

Increased color density is the combined result of more complete retention of soluble elements during initial fixation by ruthenium red and the formation of a colored reaction product between the bound ruthenium red and osmium which is further intensified by p-phenylenediamine.     

Ruthenium red staining for ultrastructural visualization of a glycoprotein layer surrounding the spore of Bacillus anthracis and Bacillus subtilis

Ruthenium red is a polycationic stain used to visualize acid polysaccharides on the outer surface of cells. Ruthenium red staining followed by electron microscopic analysis was used to demonstrate the presence of an external glycoprotein layer surrounding the spore of both Bacillus anthracis and Bacillus subtilis. This layer is less apparent with traditional staining methods used for electron microscopy. Renografin gradients were used to purify B. subtilis spores. These purified spores displayed greatly enhanced staining with ruthenium red, indicating nonspecific binding of renografin, which has a major carbohydrate constituent, methylglucamine. For B. anthracis, staining with ruthenium red was sufficiently intense that it was not significantly enhanced by renografin purification. In addition to demonstrating a previously undiscovered layer surrounding the spores of B. subtilis, the results help explain a long-standing controversy as to ultrastructural differences among these genetically closely related organisms. Ruthenium red staining provides an important addition to the identification of surface glycoproteins in studies to define similarities and differences in the exosporium layers of Bacillus species.     

Total cellular RNA content: correlation between flow cytometry and ultraviolet spectroscopy

Total RNA content in Chinese hamster ovary and HeLa-S3 cells determined by ultraviolet spectroscopy is compared with the red fluorescence distribution of acridine orange-stained cells observed by flow cytometry. A correlation coefficient of 0.93 is obtained when these methods of estimating RNA content are compared after various RNAse treatments. These data suggest that acridine orange staining effectively quantitates total cellular RNA content when analyzed by flow cytometry, although DNA is also shown to contribute a low but significant background of red fluorescence.     

Confocal Fluorescence Microscopic Observation on the Transcellular Distribution of Actin Filaments in the Epidermis of Garlic Clove Sheath

By means of paraformaldehyde fixation, Triton X-100 extraction and TRITC-phalloidin staining, the presence and distribution patterns of F-actin in the outer epidermal cells of the garlic (Allium sativum L.) sheath were studied with fluorescence probe technique and confocal laser scanning microscopy. There were a lot of actin filaments (AFs) impenetrate the cell wall, but the AFs with red fluorescence were absent when the cells were treated with cytochalasin D before fixation; the same result was obtained when the cells were treated with unlabeled phalloidin. These results indicate the presence of F-actin in the intercellular channels and that it is related to the plasmodesmata and intercellular trafficking of macromolecules.     

Electron microscopy of the cell envelope of Ferrobacillus ferrooxidans prepared by freeze-etching and chemical fixation techniques

Remsen, C. C. (Swiss Federation Institute of Technology, Zurich, Switzerland), and D. G. Lundgren. Electron microscopy of the cell envelope of Ferrobacillus ferrooxidans prepared by freeze-etching and chemical fixation techniques. J. Bacteriol. 92:1765-1771. 1966.-A comparison was made of the fine structure of the cell envelope of the gram-negative bacterium Ferrobacillus ferrooxidans when cells were prepared for microscopy by freeze-etching and chemical fixation techniques. Cell envelopes of chemically fixed cells appeared as five separate layers distinguishable by their location and electron density. Frozen-etched cells showed a three-layered complex with each layer measuring approximately 100 A in thickness. The latter technique is considered to be "artifact-free" and, as a technique, yields purely morphological information on the natural state. The three layers revealed by freeze-etching are: the outer layer, a lipoprotein-lipopolysaccharide layer; the middle layer, a layer composed of globular protein attached to fibrillar mucopeptide; and the innermost layer, the cytoplasmic membrane. The latter was covered with 100 to 120 A particles. The relationship of the aforementioned layers to those seen in chemically fixed cells is discussed.     

Mise en évidence par immunofluorescence des cellules sécrétrices de glucagon dans le pancréas endocrine du poisson téléostéen Xiphophorus helleri H.

Summary The ruthenium red staining of the surface coats was studied in the adrenal medullary cells of golden hamster. Both immersion and perfusion fixation was used with the ruthenium red containing fixative, however, only the perfusion fixation gave positive results. A rather thick electron dense ruthenium red positive layer was found on the plasma membrane of the endothelial cells, around the capillaries in the basal lamina, in the basement membrane of the chromaffin cells as well as on the apical and lateral cell surfaces of the adrenomedullary cells. Coated pits and coated vesicles usually showed an intensive ruthenium red staining, but the other cell components in the cytoplasm did not. On the basis of these observations author suggests that the ruthenium red positive material corresponds to acidic mucopolysaccharides in the hamster adrenal medulla, and its wide-ranging occurrence is indicative of its significance in the secretion process of catecholamines.Wellcome Research Fellow.     

A comparison of methods for morphological studies of cultured cells

Summary A number of fixation methods for different types of cells in culture were compared, and the best preservation of nuclear and cytoplasmic details was obtained by fixation with Bouin's solution for 15 min, prior to staining with hematoxylin and eosin. All of the fixatives, including Bouin's solution, damaged various structures, notably the peripheral glas-attached cytoplasm and the intercellular connections. Micrographs obtained by bright field, phase contrast, and interference contrast (Nomarski) microscopy are presented. Much more realistic pictures, bringing out details not observed after fixation and staining, were obtained by Nomarski microscopy of living, unfixed cultures. Most conspicuous were numerous thin, cytoplasmic, cilia-like extensions, concentrated on the glass-attached peripheral margins, which were also visible on other cell surfaces and as intercellular connections. These structures were most characteristic of SV40-transformed human amnion cells. Although fixation and staining emphasize certain cell components (for example, inclusion bodies), many aspects of cellular morphology are better demonstrated by observing living cells by interference microscopy or by Nomarski interference contrast microscopy. Surface features of unfixed cells, seen by Nomarski interference contrast microscopy, were similar to the surface features of glutaraldehyde-osmium tetroxide-fixed cells studied as metallic replicas in the electron microscope. Supported in part by National Cancer Institute Research Grant CA-08748 and contributions from the Albert Soiland Cancer Foundation.     

Ruthenium red binding to cell coat in neoplastic neurogenic cell lines in culture.

The cell coat of cultivated fetal rat brain cells as well as malignant rat neurogenic cell lines in culture were studied by transmission electron microscopy with the ruthenium red staining technique. Some of the transformed cell lines demonstrated alteration in the bindng properties of ruthenium red to the cell surface. Otherwise no significant correlation between the visualized cell coat thickness and neoplastic transformation was noted.     

Structure of Escherichia coli After Freeze-Etching

Survival of Escherichia coli, quick-frozen under conditions similar to those employed for freeze-etching, is close to 100%. For determination of cell shrinkage, the diameters of freeze-etched E. coli cells (average, 0.99 mum) were compared with those of preparations after negative staining and after ultrathin sectioning. Negatively stained cells measured from 0.65 to 1.0 mum in diameter, and ultrathin sections showed average cell diameters of 0.70 mum. Freeze-etched replicas of logarithmically growing, as well as stationary, E. coli B cells revealed a smooth, finely pitted cell surface in contrast to cell surfaces seen with other preparative methods. The frozen cell wall may cleave in two planes, exposing (i) a smooth fracture face within the lipid layer and (ii) in rare instances an ill-defined particulate layer. Most frequently, however, cleavage of the envelope occurred between wall and protoplasmic membrane; large areas of the membrane were then exposed and showed a surface studded with predominantly spherical particles, an appearance which did not significantly change when the cells were fixed in formaldehyde and osmium tetroxide before freeze-etching. The distribution of these particles differed between logarithmically growing cells and stationary cells.     

Nile red staining of lysosomal phospholipid inclusions

Summary We have employed the fluorescent dye nile red to distinguish between normal cells and cells containing lysosomal accumulations of phospholipids. When fibroblasts from an individual with a genetic deficiency in lysosomal sphingomyelinase activity (Niemann-Pick disease) were stained with nile red and visualized by fluorescence microscopy, orange-colored inclusions were observed throughout the cytoplasm. The orange fluorescent bodies could be distinguished from the neutral lipid droplets that fluoresce a brilliant yellow-gold in the presence of nile red. These inclusions were also observed in alveolar macrophages obtained from rats treated with amiodarone, an antiarrhythmic agent known to produce lysosomal phospholipidosis. Flow cytofluorometric analysis revealed that staining of these phospholipid-rich macrophages with nile red can distinguish them from control alveolar macrophages. These results demonstrate that nile red can be employed for the rapid staining of cellular phospholipid inclusions.     

Nile red staining of lysosomal phospholipid inclusions.

We have employed the fluorescent dye nile red to distinguish between normal cells and cells containing lysosomal accumulations of phospholipids. When fibroblasts from an individual with a genetic deficiency in lysosomal sphingomyelinase activity (Niemann-Pick disease) were stained with nile red and visualized by fluorescence microscopy, orange-colored inclusions were observed throughout the cytoplasm. The orange fluorescent bodies could be distinguished from the neutral lipid droplets that fluoresce a brilliant yellow-gold in the presence of nile red. These inclusions were also observed in alveolar macrophages obtained from rats treated with amiodarone, an antiarrhythmic agent known to produce lysosomal phospholipidosis. Flow cytofluorometric analysis revealed that staining of these phospholipid-rich macrophages with nile red can distinguish them from control alveolar macrophages. These results demonstrate that nile red can be employed for the rapid staining of cellular phospholipid inclusions.     

Glycoproteins in membranes of secretory granules of the anterior pituitary gland.

Rat anterior pituitary glands were examined by electron microscopy after staining with five different histochemical stains. Histochemical reactions were observed in the cell coat, cell membrane and the membrane surrounding the secretory granules in all anterior pituitary cells following staining with phosphotungstic acid (PTA), chromic acid and PTA, the periodic acid-thiosemicarbazide-silver protein method (PA-TSC-SP) of Thiéry, ruthenium red and concanavalin A. The staining was abolished when the sections were preincubated with pronase, neuraminidase or trypsin and subsequently exposed to PTA, chromic acid and PTA or PA-TSC-SP. The possible functional role of the glycoproteins present in the membrane surrounding the secretory granules is considered.     

Improved preservation of rat epididymal sperm for high-resolution low-voltage scanning electron microscopy (HR-LVSEM)

Various fixation protocols were used in an attempt to improve preservation of rat epididymal sperm for high-resolution low-voltage scanning electron microscopy (HR-LVSEM). Wash solutions and fixatives of different composition and osmolarity were tested. Paraformaldehyde and glutaraldehyde concentrations were varied between 0.5% and 3%. Ruthenium red was tested as an additive in both primary fixation and postfixation, or in postfixation alone. HR-LVSEM revealed various degrees of ruffing, folding, blebbing, and peeling off of the plasma membrane, as well as holes of different sizes. The plasma membrane overlying the acrosome and the connecting piece proved to be particularly sensitive to varying fixation conditions. Consistent topographical differences were revealed among the different domains over the sperm head. Most of the differences were considered to be artifacts. Their consistency, however, suggests that structural and biochemical differences exist either within the membrane or in the structures subjacent to the membrane. Primary fixation turned out to be less critical than postfixation. Preservation of a smooth plasma membrane without holes could only be achieved when primary fixation in low aldehyde concentrations, with or without ruthenium red, was followed by postfixation with OsO₄ and 1,000 ppm ruthenium red. Examination of thin sections of the same material confirmed that even a considerable number of small holes are difficult to detect in transmission electron microscopy. These results show that with the recent increase in resolution of LVSEM there is need for further effort to improve sample processing. © 1993 Wiley-Liss, Inc.     

Whole blood staining in suspension for nonspecific esterase and alkaline phosphatase analyzed with a Technicon H-1.

We adapted previously published methods for nonspecific esterase and alkaline phosphatase staining of white blood cells in suspension for use on a Technicon H-1 hematology analyzer. The objective was to develop a semiautomated method using whole blood that could be employed on a large scale for hematology laboratory applications, including toxicology studies, measurement of neutrophil left shift, and cytochemical classification of myeloid leukemias. The nonspecific esterase method uses the pararosaniline stain, generating the unstable substrate from two stable precursors. Whole blood is added to the substrate plus dye mix. Next, acid lysis and fixation steps destroy red cells and stabilize the monocyte staining. The alkaline phosphatase stain employs a stable naphthyl phosphate substrate and fast blue B coupling dye. The red cells are lysed with a pH 10.3 propanediol buffer, and the white blood cells are then stabilized with formalin fixation. For both methods the staining is performed off-line, and the sample is then diluted with propanediol to match the refractive index of the sheath on the H-1 analyzer, before aspiration into the direct cytometry port. A cytogram of scattered versus absorbed light is obtained. The number of cells staining and the intensity of the stain can be quantified from the cytogram.     

Electron microscopy of adhesive interactions between Gardnerella vaginalis and vaginal epithelial cells, McCoy cells and human red blood cells

Exfoliated vaginal epithelial cells with attached bacteria, termed 'clue cells', which were procured from a patient with non-specific vaginitis, were stained with ruthenium red and examined by transmission electron microscopy. The attached bacteria appeared to adhere by means of an outer fibrillar coat. An epithelial tissue culture cell line (McCoy) and human red blood cells to which strains of Gardnerella vaginalis attached were similarly examined. The adherence of G. vaginalis to the epithelial cell line appeared to be mediated by an outer fibrillar coat while adherence to red cells appeared to be mediated by fimbriae. Transmission electron microscopy was performed on the Gardnerella strains used. Thin sections of tissue-culture-adherent strains revealed a dense outer fibrillar coat whereas the surface of the haemagglutinating strains showed fine fimbriae. Negative staining of haemagglutinating strains demonstrated fimbriae on a minority of organisms.     

Glycoproteins in membranes of secretory granules of the anterior pituitary gland

Summary Rat anterior pituitary glands were examined by electron microscopy after staining with five different histochemical stains. Histochemical reactions were observed in the cell coat, cell membrane and the membrane surrounding the secretory granules in all anterior pituitary cells following staining with phosphotungstic acid (PTA), chromic acid and PTA, the periodic acid-thiosemicarbazide-silver protein method (PA-TSC-SP) of Thiéry, ruthenium red and concanavalin A. The staining was abolished when the sections were preincubated with pronase, neuraminidase or trypsin and subsequently exposed to PTA, chromic acid and PTA or PA-TSC-SP. The possible functional role of the glycoproteins present in the membrane surrounding the secretory granules is considered.