scholarly journals PRESERVATION OF THE ULTRASTRUCTURE OF BACILLUS SUBTILIS BY CHEMICAL FIXATION AS VERIFIED BY FREEZE-ETCHING

1969 ◽  
Vol 42 (3) ◽  
pp. 733-744 ◽  
Author(s):  
N. Nanninga
Keyword(s):  
Bacillus Subtilis ◽  
Plasma Membrane ◽  
Osmium Tetroxide ◽  
Structural Changes ◽  
Uranyl Acetate ◽  
Chemical Fixation ◽  
Fixation Procedure ◽  
Etching Technique ◽  
Freeze Etching

The present study on the ultrastructure of Bacillus subtilis was undertaken in order to examine by means of the freeze-etching technique possible structural changes occurring during the chemical fixation procedure (Ryter-Kellenberger (R-K) fixation). Three stages were followed by freeze-etching, viz.: (a) fixation in osmium tetroxide, (b) fixation in osmium tetroxide and posttreatment with uranyl acetate, and (c) fixation in osmium tetroxide, posttreatment in uranyl acetate, and dehydration in a graded series of acetone. Preparations were made after each stage in the presence of 20% glycerol. Good preservation of ultrastructure was observed, after any of the three treatments, of the outer surface of the plasma membrane, and the inner surface of the plasma membrane. No alteration in fracturing properties could be observed. However, if we are to judge by the results of freeze-etching, any of the successive steps of the chemical fixation procedure achieve strong contrast between the nucleoplasmic region and the cytoplasm. Dependent on the quality of fixation, very delicately preserved DNA fibrils or strongly aggregated ones were seen. It appears that R-K fixation is capable of producing more or less distinctly visible changes in the native state of the nucleoplasm in young cells of B. subtilis.

scholarly journals STRUCTURAL FEATURES OF MESOSOMES (CHONDRIOIDS) OF BACILLUS SUBTILIS AFTER FREEZE-ETCHING

1968 ◽  
Vol 39 (2) ◽  
pp. 251-263 ◽  
Author(s):  
N. Nanninga
Keyword(s):  
Cell Wall ◽  
Bacillus Subtilis ◽  
Plasma Membrane ◽  
Outer Surface ◽  
Osmium Tetroxide ◽  
Close Association ◽  
Membrane Surface ◽  
Bacterial Membranes ◽  
Inner Surface ◽  
Freeze Etching

Freeze-etched cells of Bacillus subtilis have been studied with the electron microscope. The outer surface of the plasma membrane, i.e. the side facing the cell wall, is covered with numerous granules and short strands, each measuring approximately 50 A in diameter. These strands are occasionally seen to enter the cell wall. The inner surface of the plasma membrane, i.e. the side facing the cytoplasm, appears to be sparsely dotted with small particles measuring about 50 A. The envelope of mesosomes differs from the plasma membrane. Blunt protrusions arise from its outer surface; the inner surface appears smooth. Stalked particles, as described by other investigators after negative staining with phosphotungstic acid, were not observed on any membrane surface in our material. Preparations were also made of specimens prefixed in osmium tetroxide prior to freeze-etching. Under these conditions the bacterial membranes appeared to be surprisingly well preserved. In contrast to directly frozen, unfixed cells, some osmium tetroxide-fixed preparations showed a differentiation in cytoplasm and nucleoplasm, which made it possible to observe the close association of the mesosome with the latter.

Mesosome-like Structures in Cryptococcus neoformans Studied by Chemical Fixation and by the Freeze-etching Technique

1968 ◽  
Vol 26 ◽  
pp. 80-81
Author(s):  
M. R. Edwards ◽  
S. C. Holt
Keyword(s):  
Electron Microscope ◽  
Cryptococcus Neoformans ◽  
Room Temperature ◽  
Osmium Tetroxide ◽  
Chemical Fixation ◽  
Etching Technique ◽  
Pathogenic Yeast ◽  
Thin Sections ◽  
Lead Salts ◽  
Freeze Etching

The general features of Cryptococcus neoformans, a pathogenic yeast, have been studied with the electron microscope. In the course of such a study it was noted that the plasma membrane of C. neoformans, occasionally invaginated into the cytoplasm and formed membranous organelles which resembled bacterial mesosomes. The present investigation was undertaken in order to examine such structures in detail and to compare the results from chemical fixation with those of freeze-etching.Cells were grown in Sabouraud's agar at 25-27° C for 24-48 hr and fixed with 4% glutaraldehyde in 0.15 M phosphate (Sbrensen's) buffer, at room temperature, for 2 hr; after being thoroughly washed in the buffer and post-fixed in osmium tetroxide, in the same buffer, they were dehydrated in ethyl alcohol and embedded in Epon. Thin sections were cut in a LKB microtome, double stained with uranyl and lead salts and examined in the Siemens Elmiskop IA.

Observations of thick and thin sections in a field-emission SEM

1994 ◽  
Vol 52 ◽  
pp. 1018-1019
Author(s):  
W. P. Wergin ◽  
S. Roy ◽  
E. F. Erbe ◽  
C. A. Murphy ◽  
C. D. Pooley
Keyword(s):  
Electron Microscope ◽  
Field Emission ◽  
Room Temperature ◽  
Osmium Tetroxide ◽  
Uranyl Acetate ◽  
Chemical Fixation ◽  
Thin Sections ◽  
Transmission Electron ◽  
Conventional Transmission Electron Microscope ◽  
Scanning Electron

Larvae of the nematode, Steinernema carpocapsae Weiser strain All, were cryofixed and freezesubstituted for 3 days in acetone containing 2% osmium tetroxide according to established procedures. Following chemical fixation, the nematodes were brought to room temperature, embedded in Spurr's medium and sectioned for observation with a Hitachi S-4100 field emission scanning electron microscope that was equipped with an Oxford CT 1500 Cryotrans System. Thin sections, about 80 nm thick, similar to those generally used in conventional transmission electron microscope (TEM) studies were mounted on copper grids and stained with uranyl acetate for 30 min and lead citrate for 5 min. Sections about 2 μm thick were also mounted and stained in a similar fashion. The grids were mounted on an Oxford grid holder, inserted into the microscope and onto a cryostage that was operated at ambient temperature. Thick and thin sections of the larvae were evaluated and photographed in the SEM at different accelerating voltages. Figs. 4 and 5 have undergone contrast conversion so that the images would resemble transmitted electron micrographs obtained with a TEM.

Perfusion fixation of lungs for structure-function analysis: credits and limitations

1982 ◽  
Vol 53 (2) ◽  
pp. 528-533 ◽  
Author(s):  
H. Bachofen ◽  
A. Ammann ◽  
D. Wangensteen ◽  
E. R. Weibel
Keyword(s):  
Osmium Tetroxide ◽  
Function Analysis ◽  
Transpulmonary Pressure ◽  
Uranyl Acetate ◽  
Vascular Perfusion ◽  
Lung Weight ◽  
Physiological Variables ◽  
Isotonic Buffer ◽  
Perfusion Fixation

The quality of tissue preservation in lungs fixed by vascular perfusion has been reevaluated. Excised rabbit lungs inflated to 60% of total lung capacity were perfused (zone III conditions) with different but widely used fixatives. The effects of the perfusates on pertinent physiological variables have been assessed by a continuous monitoring, the effects on the pulmonary microstructure by qualitative and morphometric analysis of electron micrographs. Important results include the following. 1) Perfusions with isotonic glutaraldehyde at flow rates within the physiological range produce large increases of perfusion pressure and lung weight that reflect intracellular, interstitial, and intra-alveolar edema. 2) No edema occurs if glutaraldehyde is added to isotonic buffer solutions (total osmolarity 510 mosM). 3) Glutaraldehyde as sole perfusate does not fully eliminate the retractive force of lung tissue. Upon release of transpulmonary pressure the lungs retract by an indeterminable amount. 4) Satisfactory results can be obtained by sequential perfusion with osmium tetroxide and uranyl acetate or glutaraldehyde (510 mosM) followed by osmium tetroxide and uranyl acetate. The latter combination yields optimal preparations to study the alveolar and capillary architecture but causes a hyperosmotic volume loss of lung cells (cell shrinkage).

scholarly journals Potassium permanganate is an excellent alternative to osmium tetroxide in freeze-substitution

2022 ◽  
Author(s):  
Martin Schauflinger ◽  
Tim Bergner ◽  
Gregor Neusser ◽  
Christine Kranz ◽  
Clarissa Read
Keyword(s):  
High Pressure ◽  
Potassium Permanganate ◽  
Lipid Membranes ◽  
Osmium Tetroxide ◽  
Freeze Substitution ◽  
Uranyl Acetate ◽  
En Bloc ◽  
Block Face ◽  
Critical Aspects

AbstractHigh-pressure freezing followed by freeze-substitution is a valuable method for ultrastructural analyses of resin-embedded biological samples. The visualization of lipid membranes is one of the most critical aspects of any ultrastructural study and can be especially challenging in high-pressure frozen specimens. Historically, osmium tetroxide has been the preferred fixative and staining agent for lipid-containing structures in freeze-substitution solutions. However, osmium tetroxide is not only a rare and expensive material, but also volatile and toxic. Here, we introduce the use of a combination of potassium permanganate, uranyl acetate, and water in acetone as complementing reagents during the freeze-substitution process. This mix imparts an intense en bloc stain to cellular ultrastructure and membranes, which makes poststaining superfluous and is well suited for block-face imaging. Thus, potassium permanganate can effectively replace osmium tetroxide in the freeze-substitution solution without sacrificing the quality of ultrastructural preservation.

scholarly journals The Striated Musculature of Blood Vessels

1959 ◽  
Vol 6 (3) ◽  
pp. 383-392 ◽  
Author(s):  
H. E. Karrer
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Electron Microscope ◽  
Osmium Tetroxide ◽  
Inferior Vena ◽  
Phosphotungstic Acid ◽  
Vena Cava ◽  
Uranyl Acetate ◽  
Transverse Tubules ◽  
Thin Sections

The musculature of small lung veins, of the thoracic portion of the inferior vena cava, and of other thoracic veins of the mouse have been studied in the electron microscope. Tissues were fixed in 1 per cent osmium tetroxide buffered with veronal, to which either sodium chloride or sucrose had been added. Methacrylate or araldite served as embedding matrices. Phosphotungstic acid or uranyl acetate was used to stain some of the preparations. Thin sections were examined in a Siemens and Halske Elmiskop Ib electron microscope. The entire musculature of the veins examined was of the striated type. It represents a variety of cardiac muscle, characterized by centrally located nuclei, typical mitochondria, and narrow I bands. Many I bands cannot be recognized at all. H and M bands are likewise indistinct. There is a double array of primary and secondary myofilaments. Mitochondria are large and numerous and contain many cristae. The endoplasmic reticulum consists of longitudinal tubules which run through the whole sarcomeres and bypass Z bands, and of transverse tubules which accompany Z bands. Some "triads," located at Z levels, consist of flattened vacuoles flanked by such transverse tubules. Small vesicles located at Z bands, close to the nucleus, and beneath the plasma membrane may represent still other portions of the reticulum.

A New Multiple Specimen Table for Freeze-Etching

1973 ◽  
Vol 31 ◽  
pp. 94-95
Author(s):  
S. K. Paik ◽  
J. R. Allen
Keyword(s):  
Electron Microscopy ◽  
Vacuum Chamber ◽  
Microscopic Image ◽  
Electron Microscopic ◽  
Etching Technique ◽  
Electron Microscopic Image ◽  
Freeze Etching ◽  
Freeze Fixation ◽  
Etched Surface

Considerable progress in freeze etching technique has been made during the last ten years. The purpose of this technique is the preparation of replicas for electron microscopy. The technique consists of four steps. After (a) freeze-fixation, (b) the specimen surface is fractured with a cooled microtome knife in the vacuum chamber, (c) etched by low temperature sublimation, and (d) a replica of the etched surface is produced by evaporation of platinum and carbon.The electron microscopic image obtained with a freeze-etched replica is highly dependent upon the quality of the replìcas. This quality is very easily affected by the thickness of the evaporated layer and the angle of shadowing.

Correlation of Ultrastructural, Morphometric and Biochemical Changes in Rats after Treatment with the Hypolipidemic Drugs Clofibrate and Probucol

1981 ◽  
Vol 39 ◽  
pp. 588-589
Author(s):  
G. E. Visscher ◽  
R. L. Robison ◽  
R. G. Engstrom
Keyword(s):  
Osmium Tetroxide ◽  
Structural Changes ◽  
Uranyl Acetate ◽  
Male Rats ◽  
Ethanol Dehydration ◽  
Sprague Dawley ◽  
Thin Sections ◽  
Biochemical Effects ◽  
Hypolipidemic Agents ◽  
Hypolipidemic Drugs

It was our goal to evaluate the reliability and reproducibility of semi-automated morphometric techniques in the analysis of structural changes observed during drug safety assessment. Studies are presented to correlate the ultrastructural, morphometric and biochemical effects that the two hypolipidemic agents, clofibrate and probucol, produce in rats.Charles River CD Sprague-Dawley derived male rats (200-225 g body wt.) were used for the three studies performed. In studies I and II, clofibrate was administered as a dietary admixture to approximate a dosage of 300 mg/kg/day for six days. In study III, clofibrate and probucol were given as dietary admixtures to approximate dosages of 300 and 250 mg/kg/day respectively for fourteen days. Processing of hepatic specimens for electron microscopy included fixation in 1.3% sym-collidine buffered osmium tetroxide, ethanol dehydration and Epon embedment. Thin sections (600Å) were stained with uranyl acetate and lead citrate. Survey and photography was performed in the manner according to Weibel. Final prints (14,400x) were analyzed with a Zeiss MOP for morphometric quantitation.

The response of cell walls of Bacillus subtilis to metals and to electron-microscopic stains

1978 ◽  
Vol 24 (2) ◽  
pp. 89-104 ◽  
Author(s):  
T. J. Beveridge
Keyword(s):  
Bacillus Subtilis ◽  
Electron Scattering ◽  
Cell Walls ◽  
Osmium Tetroxide ◽  
Uranyl Acetate ◽  
Ruthenium Red ◽  
Transition Elements ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Wall Ultrastructure

Purified cell walls of Bacillus subtilis were subjected to solutions of 40 independent metals and the metal uptake, the electron-scattering power of thin sections, and the type of staining response evaluated. This was repeated for six typical electron-microscopic stains (uranyl acetate, uranyl magnesium acetate, osmium tetroxide, Os-meth, osmium-dimethylethylenediamine, and ruthenium red) and one new staining reagent (a potassium platinum chloride – dimethylsulfoxide complex) whose specificity is for amine functions. The reaction of select metals can be specific in terms of both uptake and staining response. Of the metals studied most transition elements had a high affinity for the wall fabric and some (i.e., Sc III, most lanthanides, U IV, Zr IV, Hf IV, Fe III, Pd II, Ru III, and In III) may be suitable as contrasting agents for electron microscopy. Furthermore, when the thickness of metal-reacted walls was compared to freeze-each and ultracryotomy data, statistical-dimensional differences were commonly seen, which indicates that wall ultrastructure can be profoundly affected by the type of metal and (or) staining reagent.

scholarly journals FINE STRUCTURE IN FROZEN-ETCHED YEAST CELLS

1963 ◽  
Vol 17 (3) ◽  
pp. 609-628 ◽  
Author(s):  
H. Moor ◽  
K. Mühlethaler
Keyword(s):  
Special Kind ◽  
Microscopic Investigation ◽  
Electron Microscopic Investigation ◽  
Yeast Cells ◽  
Chemical Fixation ◽  
Membrane Structures ◽  
Electron Microscopic ◽  
Etching Technique ◽  
Cross Sectional ◽  
Freeze Etching

The freeze-etching technique, which is a special kind of freeze-drying, allows electron microscopic investigation of cells and tissues in the frozen state. In regard to yeast cells (Saccharomyces cerevisiae) a freeze-fixation technique has been developed which does not kill the object. The electron micrographs therefore are considered to impart an image of high fidelity. The cutting of the frozen object, which actually consists of a fine splintering, produces not only cross-sectional views (cross-fractures) of the structures but also surface views of the membranes and organelles. Many surface structures are described which have not been shown by the usual sectioning techniques. The cytoplasmic membrane contains hexagonal arrangements of particles which are apparently involved in the production of the glucan fibrils of the cell wall. Alterations of the distribution of nuclear pores are shown in cells of different ages. Freeze-etching enables a clear distinction of endoplasmic reticulum and vacuoles in yeast cells. The membranes of the vesicular systems are covered by ribosomes arranged in circular patterns. The mitochondrial envelope shows small perforations which could allow the exchange of macromolecules. The storage granules consist of concentric layers of lipid, presumably phosphatide. A Golgi apparatus has been detected which may be involved in the storage of lipid. The structure of the unit membrane and the membrane structures of all organelles as revealed by chemical fixation are confirmed in principle. Glycogen agglomerations are identified in the ground plasm of older cells. Insight into artifacts introduced by common chemical fixation and embedding techniques is obtained and discussed.

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