Staining of Tissue Sections for Electron Microscopy with Heavy Metals

Heavy metals may be incorporated from solution into tissue sections for electron microscopy. The resulting increase in density of the tissue provides greatly enhanced contrast with minimal distortion. Relative densities of various structures are found to depend on the heavy metal ions present and on the conditions of staining. Certain hitherto unobserved details are revealed and some sort of specificity exists, although the factors involved are not yet understood.     

Kinetics of Lead Citrate Staining of Thin Sections for Electron Microscopy

The staining of thin sections with lead citrate shows an initial increase followed by a decrease much later; the rate of the initial increase and subsequent loss varies for different cellular components. The decrease eventually reaches a stable minimum. At this level electron scattering is less than that of unstained sections, demonstrating a loss of biological material.

Lead citrate used as a poststain following uranyl acetate causes an increase in electron density that is independent of staining time over 1-30 rain; this increase appears to depend only on the quantity of uranyl acetate already bound, implying that the lead binds predominantly to the uranyl acetate.     

Silver Methenamine Staining for Scanning Electron Microscopy of Bone Sections Containing Biomaterials

Sections of tissue containing orthopedic materials are currently used to study the compatibility of those materials and to perform electron probe microanalysis at the material-tissue interface. Identification of the cells in contact with the material by Scanning electron microscopy (SEM) is of interest. We have developed a method for staining cells and tissue structures embedded in polymethyl methacrylate with silver methenamine once the sections have been obtained. Sections were prepared by grinding, and the silver methenamine was applied after oxidation with periodic acid. The procedure was carried out in a microwave oven. Backscatter SEM showed staining of the cell nucleus membrane, chromatin, the nuclear organizers, and the chromosomes of dividing cells. The cytoplasm and the cytoplasmic membrane were also stained. Collagen fibers of the extracellular matrix and the mineralized matrix of bone were labeled. Material particles in the macrophages were easily recognizable and Energy-Dispersive Spectrometer were not impaired by the presence of silver in the preparation.     

A Simple Holder for Efficient Mass Staining of Thin Sections for Electron Microscopy

An inexpensive simply-constructed gridholder for reproducible staining of sections and storage of grids is made of a platelet of dental wax (Ladd No. 2460, or Belladi, rosa, normal) mounted on a glass slide. The grids are simply inserted perpendicularly into the wax and are held securely at their edges by the slightly sticky wax. Staining solutions may be applied directly to the standing grids or the gridholder may be inverted in a trough containing the staining solution, thus avoiding excessive contact with the air. Washing is carried out by dipping the gridholder in a series of beakers with distilled water or by flushing gently. The grids may be labeled directly on the slide and are therefore identified easily. The wax-gridholder eliminates excessive handling of the grids with tweezers not only during the staining procedure but as well as during storage and thus helps to prevent mechanical damage of the delicate sections mounted on the grids. Waxes other than those specified should not be used because of a tendency for particles to adhere to the grids, with the attendant possibility of column contamination.     

Location of Polysaccharide on Chlamydia psittaci by Silver-Methenamine Staining and Electron Microscopy

Previous serological studies have indicated that the group antigen of chlamydial organisms is composed of an acidic polysaccharide and a lipid component. The present study was undertaken in an effort to locate this polysaccharide complex by use of electron microscopy and a silver-methenamine marker. The meningopneumonitis strain of Chlamydia psittaci was propagated in HeLa-M cell culture. Organisms were purified by differential centrifugation, treatment with Genetron, and by gel filtration. After fixation and embedding, sections were obtained for electron microscopy. Sections were stained for carbohydrates with silver-methenamine. A double layer of regularly spaced silver grains of uniform size was observed at the periphery of the sectioned organisms tracing the contours of the surface membrane (cell wall). This intensity of staining was observed only when sections were oxidized with periodate prior to silver-methenamine staining. Prior treatment with 1% sodium deoxycholate resulted in a significant reduction in staining. It is considered probable that the periodate-sensitive polysaccharide found at the periphery of the organisms represents, or is a component of, the group antigen of these organisms.     

Improved Fixation of Cellulose-Acetate Reverse-Osmosis Membrane for Scanning Electron Microscopy

Fixation of cellulose-acetate membranes with either glutaraldehyde-osmium tetroxide or glutaraldehyde-ruthenium tetroxide resulted in extensive electron beam damage. Beam damage was eliminated and the bacterial surface structure was preserved, however, when cellulose-acetate membranes were fixed with glutaraldehyderuthenium tetroxide and treated successively with thiocarbohydrazide and osmium tetroxide.     

Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of natural proteins have a molecular mass between 40 - 200 kDa^1,2, a robust and high-throughput method with a nanometer resolution capability is needed. Negative staining (NS) electron microscopy (EM) is an easy, rapid, and qualitative approach which has frequently been used in research laboratories to examine protein structure and protein-protein interactions. Unfortunately, conventional NS protocols often generate structural artifacts on proteins, especially with lipoproteins that usually form presenting rouleaux artifacts. By using images of lipoproteins from cryo-electron microscopy (cryo-EM) as a standard, the key parameters in NS specimen preparation conditions were recently screened and reported as the optimized NS protocol (OpNS), a modified conventional NS protocol ³ . Artifacts like rouleaux can be greatly limited by OpNS, additionally providing high contrast along with reasonably high‐resolution (near 1 nm) images of small and asymmetric proteins. These high-resolution and high contrast images are even favorable for an individual protein (a single object, no average) 3D reconstruction, such as a 160 kDa antibody, through the method of electron tomography^4,5. Moreover, OpNS can be a high‐throughput tool to examine hundreds of samples of small proteins. For example, the previously published mechanism of 53 kDa cholesteryl ester transfer protein (CETP) involved the screening and imaging of hundreds of samples ⁶. Considering cryo-EM rarely successfully images proteins less than 200 kDa has yet to publish any study involving screening over one hundred sample conditions, it is fair to call OpNS a high-throughput method for studying small proteins. Hopefully the OpNS protocol presented here can be a useful tool to push the boundaries of EM and accelerate EM studies into small protein structure, dynamics and mechanisms.     

Lead-Ammonium Acetate; A Staining Medium for Electron Microscopy Free of Contamination by Carbonate

A solution which is stable toward atmospheric CO₂ can be made by dissolving 18 gm of NH₄Ac in 100 ml of a saturated solution of PbOAc₂. Ultrathin sections stained for 45 min in the solution remain free of contamination by precipitated carbonate and show good contrast. No precautions to avoid contact with air need be taken during staining, filtration or storage. The pH of the solution is about 6.8.     

Examination of Virus-Infected Cultured Cells by Scanning Electron Microscopy

The scanning electron microscope (SEM) was used to detect changes in morphology of BSC-1 cells after infection with vesicular stomatitis virus (VSV) or herpes simplex virus. The morphological changes of the infected cells were related to the length of time of infection and to the virus used. Extensive alteration to the cytoplasm could be seen 24 and 48 hr after infection with 10 and 320 TCID(50) of VSV. Within 24 hr after infection with 1 TCID(50) of herpes simplex, a few nuclei were swollen. However, 72 hr after infection with 100 TCID(50) of herpesvirus, many nuclei were swollen and appeared in large aggregates, probably representing formation of a polykaryocyte. Corresponding samples stained with May-Grunwald-Giemsa were observed in the light microscope and morphological changes were compared to those seen with the SEM.     

Staining of Bacteriophages for Light Microscopy

Bacteriophages can be stained with a flagella stain, making them visible by light microscopy.     

A Structural Study of Isolated Mammalian Centrioles Using Negative Staining Electron Microscopy

We have used a combination of centrifugation onto electron microscope grids and negative staining to study the structure of isolated mammalian centrioles. The technique relies on visualisation of structural detail by use of a goldthioglucose negative stain. The approach provides an easy structural definition of the mature and immature centriole and has revealed some novel proximal projections on the mature centriole. The rapid technique should prove of use in future analyses of centriolar structure and biochemistry.     

用DNA银染法研究传染性软疣病毒的形态发生发育过程

用ＤＮＡ银染技术显示了传染性软疣病毒（ＭＣＶ）形态发生发育及其过程中ＤＮＡ的变化。结果显示：在被感染的皮肤表皮细胞内都有一个大小及构型不同的银染区（病毒工厂）。ＭＣＶ的发生发育均在银染区内而不在胞质区内。其发生过程是先在细胞一侧的胞质内复制合成病毒ＤＮＡ等物质,形成中等密度的银粒大小不等的银染区（病毒前包涵体区）,然后在其中形成致密的细粒状银染区（病毒前基质区）,接着在后者的周围出现弧形的粗粒银沉淀（初期ＭＣＶ的外膜）,逐渐分割包围病毒前基质而形成初期ＭＣＶ。在发育过程中,初期ＭＣＶ的外膜、基质,核心外膜及核心均经过一系列的形态变化。侧体是中期ＭＣＶ向成熟期发育中出现的暂时性结构,其本质为含ＤＮＡ成分的病毒基质。本研究提示：ＭＣＶ的ＤＮＡ物质进入皮肤表皮细胞后能大量复制,合成大量的病毒蛋白质,自主地装配成完整的初期ＭＣＶ,后者有独特的形态发育过程。     

Vital Staining by Fe and Mn Salts for Electron Microscopy of Plant Tissue (Elodea)

Electron microscopy can demonstrate Fe or Mn accumulated by living Elodea leaves from a solution containing ions of either of these two metals. The Fe and Mn accumulation is deemed to be metabolic because it does not interfere with growth or cyclosis, and because it occurs in light only-not in darkness. The metals, therefore, constitute vital stains within the leaf cells, which intensify pictures of the endoplasmic reticulum, plasmalemmae, tonoplasts, and structures of the cell walls, nuclei, chloroplasts, and mitochondria. The electron micrographs show differences between Fe and Mn distribution, apparently due to the individual biochemical behavior of the two elements. This can be demonstrated at the submicroscopic level where each is utilized as a constituent of an enzyme or in the production of an Fe or Mn containing metabolite.     

Rapid Semiquantitative Method for Screening Large Numbers of Virus Samples by Negative Staining Electron Microscopy

A rapid, semiquantitative method for screening large numbers of virus samples by negative staining electron microscopy is presented. Results obtained by this method are compared with results obtained by the pseudoreplica method and a measureddrop method. A figure is presented which represents the limit of detectability for virus by negative staining.     

Scanning Electron Microscopy of the Extracellular Matrix of Amphibian Gastrulae by Freeze-drying

In order to demonstrate the extracellular matrix (ECM) which tends to be missing from specimens prepared by ordinary procedures, a freeze-drying method was applied to gastrulae of the newt ( Cynops pyrrhogaster ) without prefixation and the embryos were examined with a scanning electron microscope (SEM). It was revealed that a prominent amount of fibrillar ECM occupied the blastocoel and amorphous ECM covered the inner surface of the blastocoelic wall (BW) as well as the surface of migrating cells. These results were compared with those obtained from observations of specimens fixed chemically.     

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.