scholarly journals Ultrastructure of microfilament bundles in baby hamster kidney (BHK-21) cells. The use of tannic acid.

1979 ◽  
Vol 80 (3) ◽  
pp. 759-766 ◽  
Author(s):  
R D Goldman ◽  
B Chojnacki ◽  
M J Yerna
Keyword(s):  
Electron Microscope ◽  
Electron Density ◽  
Tannic Acid ◽  
Osmium Tetroxide ◽  
Stress Fibers ◽  
Supramolecular Organization ◽  
Glutaraldehyde Fixation ◽  
Microfilament Bundles ◽  
Electron Density Shifts

After standard glutaraldehyde-osmium tetroxide fixation procedures, the majority of microfilament bundles in BHK-21 cells exhibit relatively uniform electron density along their long axes. The inclusion of tannic acid in the glutaraldehyde fixation solution results in obvious electron density shifts along the majority of microfilament bundles. Striated patterens are frequently observed which consist of regularly spaced electron dense (D) and electron lucid (L) bands. A striated pattern is also observed along many BHK-21 stress fibers after processing for indirect immunofluorescence utilizing BHK-21 myosin antiserum. A direct correlation of these periodicities seen by light and electron microscope techniques is impossible at the present time. However, comparative measurements indicate that the overall patterns seen in the immunofluorescence and electron microscope preparations are similar. The ultrastructural results provide an initial clue for the ultimate determination of the supramolecular organization of contracile proteins other than actin within the microfilament bundles of non-muscle cells.

scholarly journals Banding and polarity of actin filaments in interphase and cleaving cells.

1980 ◽  
Vol 86 (2) ◽  
pp. 568-575 ◽  
Author(s):  
J M Sanger ◽  
J W Sanger
Keyword(s):  
Electron Microscopy ◽  
Tannic Acid ◽  
Actin Filaments ◽  
Cell Junctions ◽  
Stress Fibers ◽  
Cleavage Furrow ◽  
Heavy Meromyosin ◽  
Glutaraldehyde Fixation ◽  
Microfilament Bundles ◽  
Uniform Polarity

Heavy meromyosin (HMM) decoration of actin filaments was used to detect the polarity of microfilaments in interphase and cleaving rat kangaroo (PtK2) cells. Ethanol at -20 degrees C was used to make the cells permeable to HMM followed by tannic acid-glutaraldehyde fixation for electron microscopy. Uniform polarity of actin filaments was observed at cell junctions and central attachment plaques with the HMM arrowheads always pointing away from the junction or plaque. Stress fibers were banded in appearance with their component microfilaments exhibiting both parallel and antiparallel orientation with respect to one another. Identical banding of microfilament bundles was also seen in cleavage furrows with the same variation in filament polarity as found in stress fibers. Similarly banded fibers were not seen outside the cleavage furrow in mitotic cells. By the time that a mid-body was present, the actin filaments in the cleavage furrow were no longer in banded fibers. The alternating dark and light bands of both the stress fibers and cleavage furrow fibers are approximately equal in length, each measuring approximately 0.16 micrometer. Actin filaments were present in both bands, and individual decorated filaments could sometimes be traced through four band lengths. Undecorated filaments, 10 nm in diameter, could often be seen within the light bands. A model is proposed to explain the arrangement of filaments in stress fibers and cleavage furrows based on the striations observed with tannic acid and the polarity of the actin filaments.

scholarly journals The ultracytochemical demonstration of a formaldehyde-resistant dehydrogenase of leuco nitroxyl analogues.

1975 ◽  
Vol 23 (11) ◽  
pp. 840-854 ◽  
Author(s):  
W A Shannon ◽  
H L Wasserkrug ◽  
R E Plapinger ◽  
D Lynm ◽  
A M Seligman
Keyword(s):  
Skeletal Muscle ◽  
Electron Microscope ◽  
Dehydrogenase Activity ◽  
Electron Density ◽  
Osmium Tetroxide ◽  
Tetrazolium Salt ◽  
Hydrogen Ions ◽  
Tetrazolium Chloride

A dehydrogenase which is relatively stable in formaldehyde fixative is demonstrated ultracytochemically by the reduction of various leuco nitroxyl analogues in rat hepatic, renal, myocardial, skeletal muscle and prostatic tubuloalveolar glandular tissues. The nonosmiophilic tetrazolium salt, t-(2'-benzothiazolyl)-5-styryl-3-(4'-phtalhydrazidyl) tetrazolium chloride, is subsequently reduced to an insoluble osmiophilic formazan by the hydrogen ions resulting from the dehydrogenase activity. Exposure of the formazan to osmium tetroxide results in electron density enabling visualization of the reaction product in the electron microscope. Known inhibitors of various dehydrogenases were utilized in an attempt to determine the existence and/or extent of any specific characteristics of the dehydrogenase(s) involved.

Lipid in Erysiphe graminis hordei and its possible role during germination

1970 ◽  
Vol 16 (11) ◽  
pp. 1041-1044 ◽  
Author(s):  
W. E. McKeen
Keyword(s):  
Fine Structure ◽  
Electron Microscope ◽  
Osmium Tetroxide ◽  
Erysiphe Graminis ◽  
Glutaraldehyde Fixation ◽  
Sudan Black B ◽  
Erysiphe Graminis Hordei ◽  
Sudan Iv ◽  
Mother Cells ◽  
Hyphal Tip

Osmiophilic bodies appear in parts of the colonial growth of Erysiphe graminis DC. f. sp. hordei Em Marchal culture CR3 growing on the susceptible commercial Keystone variety of barley. They are readily observed by the light and electron microscope after osmium tetroxide staining and are abundant in conidiophores, conidia, and mycelium except in haustorial mother cells, in which they are usually absent. The metabolism of haustorial mother cells is distinct and the fine structure of adjoining cells is frequently different. Osmiophilic bodies are absent from the growing hyphal tip, but gradually increase in number and size further back in the terminal cell. Electron micrographs show that they are intracytoplasmic, intravacuolar, and up to 1 μ in diameter. When the colony is washed with acetone or alcohol rather than with aqueous buffer, after glutaraldehyde fixation, before osmium tetroxide fixation, the osmiophilic bodies are removed, indicating that they are lipids. Fat stains, Sudan black B, and Sudan IV stain these bodies. Perhaps the water needs of the germinating conidium are met in part by the oxidation of fats.

On the variability of spherosome-like bodies in Phaseolus vulgaris

1970 ◽  
Vol 48 (8) ◽  
pp. 1477-1480 ◽  
Author(s):  
A. K. Mishra ◽  
J. Ross Colvin
Keyword(s):  
Electron Microscope ◽  
Phaseolus Vulgaris ◽  
Electron Density ◽  
Potassium Permanganate ◽  
Osmium Tetroxide ◽  
Marked Variation ◽  
Phaseolus Vulgaris L ◽  
Shoot Apices ◽  
Distribution Shape ◽  
Physiological Heterogeneity

Root and shoot apices and primary leaves of 3- to 4-day-old seedlings of Phaseolus vulgaris L. were examined in the electron microscope after fixation with (a) glutaraldehyde followed by osmium tetroxide, (b) glutaraldehyde followed by potassium permanganate, (c) osmium tetroxide alone, and (d) potassium permanganate alone. The distribution, shape, and substructure of the spherosome-like bodies in the cytoplasm varied markedly depending upon the tissue, its age, and the fixation used. Shapes varied from stellate through polygonal to spherical. The electron density of the interior of the organelles varied from uniformly dark after osmium tetroxide fixation to almost none after permanganate fixation. In addition to differences caused by fixatives, marked variation of the electron density of the same organelles with identical fixation was observed. Structural, and possibly physiological, heterogeneity seems to be characteristic of spherosomes but in spite of their variability, they may be recognized in the electron microscope as a group of similar structures distinct from other organelles.

Fixation and conductive staining by tannin-RuO4 for transmission and scanning electron microscopy

1990 ◽  
Vol 48 (3) ◽  
pp. 24-25
Author(s):  
Gen Takahashi
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Thin Section ◽  
Tannic Acid ◽  
Osmium Tetroxide ◽  
Glutaraldehyde Fixation ◽  
Ruthenium Tetroxide ◽  
Cellular Ultrastructure ◽  
Scanning Electron

Ruthenium tetroxide(RuO4) was first used as a stain in histology by Ranvier in 1887. Its use in TEM as a fixative or stain was reported by Gaylarde et al. However,the preservation of the cellular ultrastructure was very poor after RuO4 fixation alone, because RuO4 is a far more vigorous oxidant than osmium tetroxide. Peltari et al reported that a preceeding glutaraldehyde fixation helped to stabilize the ultrastructure, although the penetration of RuO4 into the tissue was poor.In the present study, RuO4 has been found to be a useful fixative and staining reagent with the prerequisite of using RuO4 as a postfixative after prefixation with tannic acid (TA)-glutaraldehyde(GL) for thin-section TEM(TA-RuO4 method) or after preceeding osmication followed by TA mordanting for non-coating SEM(OsO4-TA-RuO4 method).TA-RuO4 method for thin-section TEM

scholarly journals FURTHER ELECTRON MICROSCOPE STUDIES ON FIBRILLAR ORGANIZATION OF THE GROUND CYTOPLASM OF CHAOS CHAOS

1968 ◽  
Vol 38 (1) ◽  
pp. 40-50 ◽  
Author(s):  
Vivianne T. Nachmias
Keyword(s):  
Electron Microscope ◽  
Alcian Blue ◽  
Room Temperature ◽  
Osmium Tetroxide ◽  
Negative Staining ◽  
Glutaraldehyde Fixation

Further evidence for fibrillar organization of the ground cytoplasm of Chaos chaos is presented. Fixations with osmium tetroxide at pH 6 or 8 and with glutaraldehyde at pH 6 or 7 were used on two preparations: (a) single actively streaming cells; (b) prechilled cells treated with 0.05% Alcian blue in the cold and returned to room temperature for 5–10 min. In addition, a 50,000 g pellet of homogenized cells was examined after fixation with glutaraldehyde-formaldehyde alone. In sections from actively streaming cells considerable numbers of filaments were observed in the uroid regions after glutaraldehyde fixation, whereas only traces of filaments were seen after osmium tetroxide fixation at either pH 6 or 8. Microtubules were not seen. In sections from dye-treated cells, filaments (4–6 mµ) and fibrils (12–15 mµ) were found with all three fixatives. The 50,000 g pellet was heterogeneous but contained both clumps of fibrils and single thick fibrils like those seen in the cytoplasm of dye-treated cells. Many fibrils of the same dimensions (12–15 mµ wide, 0.5 µ long) were also seen in the supernatant above the pellet. Negative staining showed that some fibrils separated into at least three strands of 4–6 mµ filaments.

scholarly journals PRESERVATION OF MYELIN LAMELLAR STRUCTURE IN THE ABSENCE OF LIPID

1967 ◽  
Vol 34 (3) ◽  
pp. 817-826 ◽  
Author(s):  
Leonard Napolitano ◽  
Francis Lebaron ◽  
Joseph Scaletti
Keyword(s):  
Fatty Acids ◽  
Liquid Chromatography ◽  
Fine Structure ◽  
Electron Microscope ◽  
Lamellar Structure ◽  
Osmium Tetroxide ◽  
Gas Liquid Chromatography ◽  
Glutaraldehyde Fixation ◽  
Thin Sections ◽  
Sciatic Nerves

The fine structure of myelin was studied in glutaraldehyde-fixed rat sciatic nerves depleted of lipid by acetone, chloroform:methanol (2:1 v/v), and chloroform:methanol:concentrated HCl (200:100:1, v/v/v). One portion of each of these nerves, plus the extracts, was saponified and analyzed by gas-liquid chromatography for fatty acids. The remainder of each nerve was stained in osmium tetroxide in CCl4 (5g/100cc) and was embedded in Epon 812. Thin sections, examined in the electron microscope, revealed the preservation of myelin lamellar structure with a 170 A periodicity in nerves depleted of 98% of their lipids. Preservation of myelin lamellar structure depended on glutaraldehyde fixation and the introduction of osmium tetroxide in a nonpolar vehicle (CCl4) after the lipids had been extracted. It is concluded that the periodic lamellar structure in electron micrographs of myelin depleted of lipid results from the complexing of osmium tetroxide, plus uranyl and lead stains, with protein.

The direct visualization of macromolecular complexes in redundant cytomembrane systems with particular reference to peripheral nerve myelin

1984 ◽  
Vol 42 ◽  
pp. 24-27
Author(s):  
Daniel C. Pease
Keyword(s):  
Peripheral Nerve ◽  
Tannic Acid ◽  
Osmium Tetroxide ◽  
Membrane System ◽  
Direct Visualization ◽  
Glutaraldehyde Fixation ◽  
Macromolecular Complexes ◽  
Cadmium Ions ◽  
Integral Proteins

A combination of little used preparative techniques permits the visualization of macromolecular complexes in peripheral nerve myelin. They can be employed effectively as well for similar particulate recognition in at least some other redundant cytomembrane systems, i.e. retinal disc membranes. The cores of the globular structures are thought to be the integral proteins of the membrane system, in glutaraldehyde-fixed myelin probably associated with phospholipid annuli.Fixation is limited largely to aldehydes, with osmium tetroxide being particularly contraindicated. Cadmium ions, which form water-insoluable salts with lecithins, seem to offer some advantages when included with a glutaraldehyde fixation. Tannic acid as a secondary fixative has proven necessary when embedments are to be avoided.

Further Observations on the Virus of Epizootic Diarrhea of Infant Mice. An Electron Microscopic Study

1967 ◽  
Vol 25 ◽  
pp. 110-111
Author(s):  
W. G. Banfield ◽  
G. Kasnic ◽  
J. H. Blackwell
Keyword(s):  
Electron Microscope ◽  
Infected Cell ◽  
Microscopic Study ◽  
Intestinal Epithelium ◽  
Ultrastructural Study ◽  
Osmium Tetroxide ◽  
Lead Citrate ◽  
Electron Microscopic ◽  
Virus Particles ◽  
Infected Cells

An ultrastructural study of the intestinal epithelium of mice infected with the agent of epizootic diarrhea of infant mice (EDIM virus) was first performed by Adams and Kraft. We have extended their observations and have found developmental forms of the virus and associated structures not reported by them.Three-day-old NLM strain mice were infected with EDIM virus and killed 48 to 168 hours later. Specimens of bowel were fixed in glutaraldehyde, post fixed in osmium tetroxide and embedded in epon. Sections were stained with uranyl magnesium acetate followed by lead citrate and examined in an updated RCA EMU-3F electron microscope.The cells containing virus particles (infected) are at the tips of the villi and occur throughout the intestine from duodenum through colon. All developmental forms of the virus are present from 48 to 168 hours after infection. Figure 1 is of cells without virus particles and figure 2 is of an infected cell. The nucleus and cytoplasm of the infected cells appear clearer than the cells without virus particles.

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