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.     

An optimized device for staining electron microscopy grids

Proper staining of grids is critical for transmission electron microscopy (TEM). Staining must be done as quickly as possible using minimal reagents and with consideration for the environment. We developed a new device for efficient staining of multiple TEM grids. We studied reagent evaporation, rinsing volume, flow rate and re-use of uranyl acetate, and provide here a procedure for efficient staining using the new device. Our device permits TEM grids to be stained with less reagent than alternative staining apparatuses; staining requires a total volume of 260 μl for five grids. Reagent evaporation is less than 6% even if used at 37° C. Moreover, our staining apparatus reduces chemical waste and shortens experiment time by staining several grids simultaneously. Our staining device is a compromise between time-consuming single grid processing and expensive commercial devices that consume large amounts of reagents.     

Improved Staining Boxes for Fast, Uniform Staining of Ultrathin Sections on Grids

A standard LKB (LKB-Produkter Ab. S161, 45 Bromma 1, Sweden) grid storage box is converted into several grid staining boxes by sawing the body of the box into segments along rows of its grid storage cavities. the staining boxes can be cut out to any required size or shape. the polymethacrylate storage box cover is discarded. Covers for the staining boxes are cut from thin sheet vinyl, which is more chemically resistant thin polymethacrylate. Corresponding 2 mm diameter holes are drilled through the vinyl covers and the bottoms of the grid storage cavities of the staining boxes to convert the storage cavities into staining chambers. for staining, the covers are tied to the boxes with sewing thread and the assembled units are put into vials. the separate staining chambers prevent intermingling of and mechanical damage to grids during the staining procedure. Ultrathin sections are more cleanly and uniformly stained in bulk by the use of these staining boxes than they are when stained individually by a standard method.     

Improved staining boxes for fast, uniform staining of ultrathin sections on grids.

A standard LKB (LKB-Produkter Ab. S-161, 25 Brommma 1, Sweden) grid storage box is converted into several grid staining boxes by sawing the body of the box into segments along rows of its grid storage cavities. The staining boxes can be cut out to any required size or shape. The polymethyacrylate storage box cover is discarded. Covers for the staining boxes are cut from thin sheet vinyl, which is more chemically resistant than polymethyacrylate. Corresponding 2 mm diameter holes are drilled through the vinyl covers and the bottoms of the grid storage cavities of the staining boxes to convert the storage cavities into staining chambers. For staining, the covers are tied to the boxes with sewing thread and the assembled units are put into vials. The separate staining chambers prevent intermingling of and mechanical damage to grids during the staining procedure. Ultrathin sections are more cleanly and uniformly stained in bulk by the use of these staining boxes than they are when stained individually by a standard method.     

An efficient staining method for ultrathin sections.

A staining method to handle simultaneously as many as 20 electron microscope grids is described. The devices used are easily constructed of readily obtained inexpensive materials. The volumes of stain and wash water required are very small and drying grids is simplified.     

An Efficient Staining Method for Ultrathin Sections

A staining method to handle simultaneously as many as 20 electron microscope grids is described. The devices used are easily constructed of readily obtained inexpensive materials. The volumes of stain and wash water required are very small and drying grids is simplified.     

Lead asparate, an en bloc contrast stain particularly useful for ultrastructural enzymology

Lead aspartate is a new en bloc stain for electron microscopy. Its predictable staining depends on chelation that results from the interaction of the two stain components, lead nitrate and aspartic acid, which must be present in a specific ratio. Lead aspartate stain is 0.02 M in lead nitrate and 0.03 M in aspartic acid, adjusted to pH 5.5. Cells or tissues are stained at 60 degrees C for 30 to 60 min. Cells stained en bloc with lead aspartate closely resemble cells stained on grids by lead citrate, except that the former seldom have contamination. En bloc staining with lead aspartate bypasses the grid-staining step so that samples can be viewed and photographed immediately after they are thin-sectioned. The lower pH of the lead aspartate solution allows counterstaining of enzyme reaction products that dissolve in the highly alkaline lead citrate stain. Lead aspartate en bloc staining to enhance contrast should especially benefit studies of ultrastructure requiring a clean and predictably lead stain.     

Intensive drying of grids after lead citrate staining lowers precipitate contamination on the surface of ultrathin sections

Two techniques are proposed to minimize precipitate contamination of ultrathin sections after lead citrate staining. According to the first one, stained and washed grids are placed, sections downward, on a stack of filter papers. The second one involves a consecutive washing of grids with distilled water, 96% ethanol, and n-hexane. Both techniques are equally efficient; the former being simpler in processing, while the latter is superior in the reproducibility of results.     

Simultaneous visualization of LDL receptor distribution and clathrin lattices on membranes torn from the upper surface of cultured cells

We have developed a method for simultaneous visualization by electron microscopy of both the distribution of cell surface receptors and architectural features of the inner membrane surface, such as clathrin-coated pits. Electron microscope grids were covered with formvar and coated with poly-L-lysine. These grids were then placed on a piece of buffer-impregnated cellulose acetate membrane filter maintained at 4 degrees C on an ice bath. Cells of interest were grown on glass coverslips and incubated with either a ligand-gold or an antibody-gold conjugate specific for the membrane determinant of interest. The coverslip with gold-labeled cells was then overlaid on the grids and pressure was applied. When the grid was removed, large areas of the upper cell surface, which had labeled determinants, remained adherent to the formvar support. With the proper staining, both the gold particles and internal membrane features could be seen at the same time in the electron microscope. This method is rapid, does not require extensive experience with electron microscopic technique, and permits viewing of membrane samples that are large enough to perform quantitative analysis of gold distribution in relation to membrane specializations.     

Structure of adsorbed fibrinogen obtained by scanning force microscopy

It is shown that scanning force microscopy (SFM), operated in the attractive mode, can be used to obtain high resolution pictures of adsorbed fibrinogen molecules on solid surfaces, without the need for staining or special microscope grids. SFM also reveals the three-dimensional structure of the adsorbed molecules. Two forms of adsorbed fibrinogen are demonstrated on hydrophobic silicone dioxide surfaces; a trinodular about 60 nm long and a globular with about a 40 nm diameter. Polymeric networks formed after storage of the surface with adsorbed fibrinogen in PBS for 11 days are also shown. The SFM-results for the trinodular structure suggest the existence of loops or peptide chains extending outside the basic structure of the fibrinogen molecule.     

Scaffold-like structures in mouse chromosomes revealed by restriction endonuclease digestion and electron microscopy

A scaffold-like structure is observed under the electron microscope when mouse chromosomes are digested with the restriction endonuclease Hae III. This structure, located in the inner part of chromatids, may correspond to those fragments of chromatin loops anchored to the chromosome scaffold and is obtained when chromosomes are treated either in suspension or attached to grids. The width of the structure is correlated with the extent of digestion in chromosomes treated in suspension. Those treated on grids show this structure whenever chromatids do not collapse. These results agree with the model of chromosome organization based on a non-histone protein scaffold.     

Morphology of sodium deoxycholate-solubilized apolipoprotein B-100 using negative stain and vitreous ice electron microscopy

The primary and secondary structures of apolipoprotein B-100 (apoB-100) are well established. Previous morphological studies have suggested that apoB is a long, flexible, threadlike molecule that encircles the low density lipoprotein (LDL) particle. Several large domain regions of the protein have been observed in frozen hydrated LDL and may be involved in anchoring of the protein to the lipid surface of LDL. Calorimetric studies of sodium deoxycholate (NaDC)-solubilized apoB indicated a similar number of independently melting domains. We therefore undertook a morphological study of NaDC-solubilized apoB-100 using negative stain and vitreous ice cryoelectron microscopy, a nonperturbing preservation technique. Negative staining experiments were performed in two ways: 1) grids were pulled through NaDC-containing buffer surfaces on which monolayers of apoB had been promoted, or 2) apoB molecules were allowed to diffuse onto carbon surfaces of grids that were floated on sample droplets. Vitrified molecules of apoB were obtained by plunging a thin fluid layer of protein adhered to a holey carbon-coated grid into supercooled ethane and by preserving the molecules in liquid nitrogen. The majority of molecules prepared in negative stain and vitreous ice were curved or arced and had alternating thin and thick regions. In negative stain, the apoB molecules lay on the grid perpendicular to the electron beam and had a mean length of 650 A. In vitreous ice the molecules were randomly oriented and their images ranged from 160 to 650 A in length. Vitrified molecules provided visualization of one or two beaded regions. Similar regions were observed in negative stain but the overall thickness was two to three times greater. Some vitrified molecules contained ribbon-like portions.Our study supports previously obtained data on molecule length but suggests that negative staining overestimates molecule width. These first images of vitrified NaDC-solubilized apoB-100 confirm the long, flexible, beaded thread morphology of the molecule and support the unique potential of this technique when coupled with proper molecule orientation and antibody labeling to correlate the tertiary structure of apoB seen in the intact particle with that of the isolated molecule.     

Method for Staining and Washing Thin Sections on Support Films

A procedure is described for staining large numbers of thin sections on support films for use with one-hole grids. The film is picked up, carried and protected using easily made plastic blocks. Loop-tipped forceps are then used to transfer tissue ribbons from the knife boat to the support film. A large number of tissue sections can then be stained and washed simultaneously in a modified Pyrex dish without damaging the film. After staining, the slot in the one-hole grid is centered over the tissue ribbon, and the grid is attached to the film. The method is suitable for serial reconstruction and the unobstructed viewing of large thin sections in the TEM.     

Nickel Grids Cause Astigmatism in the Transmission Electron Microscope

Nickel grids are used in various methods (e.g., in immunocytochemistry) where chemically inert grids are required. Recently (Neiss 1983) we have used formvar-coated nickel grids when removing osmium from mounted ultrathin sections with 10% periodic acid (Lewis and Knight 1977). This is possible because nickel, unlike copper, adequately withstands the oxidation necessary for osmium removal. Handling sections on nickel grids avoids the disadvantages of free-floating sections, whose use for osmium removal has been recommended by Lewis and Knight (1977, chapter 2.2.3).     

Preparing Mixed Cellulose Ester or Polycarbonate Membrane Filter Replicas for Transmission Electron Microscopy

Simple methods for preparing large numbers of grids exhibiting excellent coverage of intact replicas on mixed cellulose ester or polycarbonate membrane filters are described. The techniques ensure that grids and carbon replicas receive identical treatment and are not rearranged or lost during processing. The techniques permit grids and filter sections to be handled en masse rather than individually. Also, replica section squares remain centered on the grids. A temporary grid storage method (“grid- pad”) is also described, which facilitates grid identification and handling.     

A search for differential polypeptide synthesis throughout the cell cycle of HeLa cells

The fine structure of resting and activated platelets was compared using two approaches novel to this dense cytoplasm. First, rapid lysis of platelets on carbon-coated grids was following by negative staining of the "cytoskeleton." Second, a brief, minimal fixation of platelets in plasma was coupled with partial lysis and examination of the unstained whole mounts at 200 kV. The results showed that the dense ground cytoplasm of discoid, fully resting platelets appeared granular or amorphous, and microfilaments were not observed. A coiled microtubule terminated in one, free, straight end. When any slight degree of activation occurred, microfilaments could be detected in the platelets. In fully spread specimens, the amorphous character of the resting cytoplasm was strikingly altered into an interconnected network of microfilaments. Stereo views of the whole mounts showed that dense granules, 100-250 nm in diameter, appeared as if suspended in the filament nets. The results support the view that platelet activation involves a major assembly of microfilaments from amorphous precursors. The change can only be seen convincingly when stringent precautions are taken during preparation because the platelets are very easily activated by thermal or mechanical stimuli.     

Sedimentation counting and morphology of Mycoplasma

Clark, Harold W. (The George Washington University School of Medicine, Washington, D.C.). Sedimentation counting and morphology of Mycoplasma. J. Bacteriol. 90:1373-1386. 1965.-The sedimentation technique for counting viral particles was applied to the quantitation and morphological identification of Mycoplasma in broth cultures. Mycoplasma, apparently in their native form, firmly adhered to the surface, when sedimented on glass cover slips or onto electron microscope grids. The sedimented cover slip preparations stained with crystal violet could be readily counted in the light microscope. The cultures sedimented onto electron microscope grids were readily counted at low magnification and provided excellent preparations for morphological examination at higher magnifications. It was found that air-dried Mycoplasma particles were enlarged considerably because of excessive flattening. Fixation of sedimented Mycoplasma particles in diluted OsO(4) prior to air drying yielded a more realistic morphology, with various sizes and shapes in the stages of the growth cycle exhibited. A new technique of differentially staining Mycoplasma colonies on agar plates was developed to facilitate the quantitation of viable colony-forming units for comparison with total counts. The use of plastic or Parafilm gaskets for dry mounting was developed to facilitate the handling and examination of the stained cover slip preparations. The results of this investigation indicated that the growth cycle of some Mycoplasma species includes a stage of hexadic fission with the cleavage of minimal reproductive units (less than 100 mmu) containing a limited deoxyribonucleic acid genetic coding molecule (approximately 4 x 10(6)).     

Improved staining of negative binding sites with ruthenium red on cryosections of frozen cells

Hexavalent cationic dye ruthenium red (RR) binds to anionic sites of cellular components, predominantly to the surface coat rich in glycoconjugates, and can be used as a marker of negative binding sites. Due to limited penetration of RR only superficial layers of cells are stained satisfactorily. To improve RR staining of L1210 leukemic cells isolated from culture and concentrated by centrifugation, cryosections of frozen cells were treated by RR to expose simultaneously all the cells and their components to the dye treatment. Cells were fixed with 2% glutaraldehyde in cacodylate buffer (CB), soaked in 2.2 mol/l sucrose and frozen by plunging into liquid nitrogen. Ultrathin cryosections were cut at a temperature of -90 degrees C, transferred to Formvar coated copper grids, postfixed with 1% OsO4 and stained with 0.05% RR in CB for 60-120 min. After removing RR solution with filter the grids were dried and examined electron microscopically. The resulting staining was a combination of a negative contrast (the plasma membrane and membranes of intracellular organelles) and of a positive contrast (cytoplasmic matrix and the extracellular coat). RR staining of negative binding sites on cryosections has proved useful for uniform exposure of all cells and cellular compartments to the dye and especially of external coat containing glycoconjugates.     

MICROGEOGRAPHIC VARIATION IN MITOCHONDRIAL DNA OF MEADOW VOLES (MICROTUS PENNSYLVANICUS) IN RELATION TO POPULATION DENSITY

We examined mitochondrial-DNA (mtDNA) sequence heterogeneity on four adjacent trapping grids in an island population of meadow voles (Microtus pennsylvanicus) at two different population densities. Four restriction endonucleases revealed 20 different mtDNA composite phenotypes in samples totaling 198 meadow voles. There were significant heterogeneities in the distribution of four common mtDNA composite phenotypes among the four trapping grids, suggesting that there is population subdivision on a fine scale. Genetic distances between grids, mtDNA diversity within grids, and G_ST also varied during the study period. We found a decrease in genetic distance and an increase in diversity when the population density was high and vice versa when the population density was low. When population density was high, the coefficient of gene differentiation was smaller than the same coefficient observed when the population density was low. These changes in population subdivision and diversity are consistent with theoretical expectations of population structure in which effective female population size and dispersal are the critical variables. The data also support the hypothesis of maintenance of mtDNA diversity by population subdivision, rapid population growth rate, and dispersal.