The effect of chemical fixatives on cell walls of Bacillus subtilis

1978 ◽  
Vol 24 (12) ◽  
pp. 1439-1451 ◽  
Author(s):  
T. J. Beveridge ◽  
F. M. R. Williams ◽  
J. J. Koval
Keyword(s):  
Bacillus Subtilis ◽  
Wall Thickness ◽  
Metal Binding ◽  
Cell Walls ◽  
Osmium Tetroxide ◽  
Binding Capacity ◽  
Statistical Technique ◽  
Uranyl Acetate ◽  
Fixation Time ◽  
Thin Sections

Cell walls of Bacillus subtilis were treated with several chemical fixatives which are commonly used preparatory to electron microscopy; i.e., osmium tetroxide, formaldehyde, acrolein, crotonaldehyde, and glutaraldehyde. Dimensional analysis was performed on thin sections of fixed walls from plastic embeddings and, by means of the statistical technique of multiple comparisons, significant differences were found between wall thicknesses from the various fixations. These differences varied with the fixation time and the type of fixative used in the reaction. When compared to embedded walls which had been stained before fixation, the overall effect was a reduction in wall thickness which was attributed to fixative action and not to the embedding or staining processes. The reduction of wall thickness was even more apparent when dimensions of fixed walls were compared to published dimensions of both frozen sections and freeze-etch profiles.Since these fixatives bind to reactive sites within the wall fabric, a change in electrochemical charge density is effected which can be monitored in terms of heavy-metal-binding capacity. Most monoaldehyde fixatives and osmium tetroxide render the wall as reactive, or less reactive, to uranyl acetate as unfixed walls, whereas glutaraldehyde can significantly increase the binding capacity.

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.

Membranous alterations in the cytoplasm and nucleus in a primitive neuroectodermal tumor of the brain

1978 ◽  
Vol 36 (2) ◽  
pp. 628-629
Author(s):  
C. N. Sun ◽  
C. Araoz ◽  
H. J. White
Keyword(s):  
Nuclear Envelope ◽  
Tumor Cells ◽  
Osmium Tetroxide ◽  
Primitive Neuroectodermal Tumor ◽  
Uranyl Acetate ◽  
Neuroectodermal Tumor ◽  
Annulate Lamellae ◽  
Lead Citrate ◽  
Thin Sections ◽  
The Brain

The ultrastructure of a cerebral primitive neuroectodermal tumor has been reported previously. In the present case, we will present some unusual previously unreported membranous structures and alterations in the cytoplasm and nucleus of the tumor cells.Specimens were cut into small pieces about 1 mm3 and immediately fixed in 4% glutaraldehyde in phosphate buffer for two hours, then post-fixed in 1% buffered osmium tetroxide for one hour. After dehydration, tissues were embedded in Epon 812. Thin sections were stained with uranyl acetate and lead citrate.In the cytoplasm of the tumor cells, we found paired cisternae (Fig. 1) and annulate lamellae (Fig. 2) noting that the annulate lamellae were sometimes associated with the outer nuclear envelope (Fig. 3). These membranous structures have been reported in other tumor cells. In our case, mitochondrial to nuclear envelope fusions were often noted (Fig. 4). Although this phenomenon was reported in an oncocytoma, their frequency in the present study is quite striking.

Ultrastructure of myoepithelial cell in parotid gland

1988 ◽  
Vol 46 ◽  
pp. 342-343
Author(s):  
C. N. Sun
Keyword(s):  
Parotid Gland ◽  
Osmium Tetroxide ◽  
Individual Cell ◽  
Branching Processes ◽  
Muscle Cells ◽  
Myoepithelial Cells ◽  
Uranyl Acetate ◽  
Thin Sections ◽  
Gland Carcinoma ◽  
Parotid Gland Carcinoma

Myoepithelial cells have been observed in the prostate, harderian, apocrine, exocrine sweat and mammary glands. Such cells and their numerous branching processes form basket-like structures around the glandular acini. Their shapes are quite different from structures seen either in spindleshaped smooth muscle cells or skeletal muscle cells. These myoepithelial cells lie on the epithelial side of the basement membrane in the glands. This presentation describes the ultrastructure of such myoepithelial cells which have been found also in the parotid gland carcinoma from a 45-year old patient.Specimens were cut into small pieces about 1 mm3 and immediately fixed in 4 percent glutaraldehyde in phosphate buffer for two hours, then post-fixed in 1 percent buffered osmium tetroxide for 1 hour. After dehydration, tissues were embedded in Epon 812. Thin sections were stained with uranyl acetate and lead citrate. Ultrastructurally, the pattern of each individual cell showed wide variations.

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.

scholarly journals Differences in cell wall of thin and thick filaments of cyanobacterium Aphanizomenon gracile SAG 31.79 and their implications for different resistance to Daphnia grazing

2016 ◽  
Author(s):  
Lukasz Wejnerowski ◽  
Slawek Cerbin ◽  
Maria K. Wojciechowicz ◽  
Marcin K. Dziuba
Keyword(s):  
Cell Wall ◽  
Wall Thickness ◽  
Cell Walls ◽  
Cell Wall Thickness ◽  
Filamentous Cyanobacterium ◽  
Thin Sections ◽  
Thick Filaments ◽  
Transmission Electron ◽  
The Difference ◽  
Aphanizomenon Gracile

<p>Recent studies have shown that the filamentous cyanobacterium <em>Aphanizomenon gracile</em> Lemmermann, strain SAG 31.79, consists of two types of filaments that differ in thickness. These two types are known to vary in resistance to <em>Daphnia</em> <em>magna</em> grazing: thin filaments (&lt;2.5 µm) are more vulnerable to grazing than the thick ones (&gt;2.5 µm). In this study, we investigated whether the difference in the vulnerability to grazing of thin and thick filaments is a result of different thickness of their cell walls, a filament stiffness determinant. We expected thick filaments to have thicker cell walls than the thin ones. Additionally, we analysed whether cell wall thickness correlates with filament thickness regardless of the filament type. A morphometric analysis of cell walls was performed using transmission electron micrographs of ultra-thin sections of the batch-cultured cyanobacterial material.  Our study revealed that the thin type of filaments had thinner cell walls than the thick filaments. Moreover, cell wall thickness was positively correlated with filament thickness. TEM (transmission electron microscopy) observations also revealed that the thin type of filaments was often at different stages of autocatalytic cell destruction, which was mainly manifested in the increase in cell vacuolization and degradation of the cytoplasm content. Based on our findings, we assume that previously reported higher resistance of thick filaments to <em>Daphnia</em> grazing results from greater stiffness and excellent physiological conditions of thick filaments. </p>

An Electron Microscopic Investigation of the Pathogenesis of Hemorrhage Induced by Rattlesnake Venom

1974 ◽  
Vol 32 ◽  
pp. 56-57
Author(s):  
Charlotte L. Ownby ◽  
Robert A. Kainer ◽  
Anthony T. Tu
Keyword(s):  
Phosphate Buffer ◽  
Osmium Tetroxide ◽  
Microscopic Investigation ◽  
Uranyl Acetate ◽  
Electron Microscopic Investigation ◽  
Lead Citrate ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Rattlesnake Venom ◽  
Diamondback Rattlesnake

One of the significant changes induced by the injection of rattlesnake (Crotalidae) venom is hemorrhage. Since crotaline antivenin does not prevent such local tissue damage, a more effective treatment of snakebite is needed. To aid in the development of such a treatment the pathogenesis of venom-induced hemorrhae was investigated.Swiss-Webster white mice were injected intramuscularly with Western diamondback rattlesnake (Crotalus atrox) venom. Two minutes after the injection, muscle tissue was obtained by bioosy from the thigh and fixed in 6% glutaraldehyde in Milloniq's phosphate buffer (DH 7.4, 2 hrs., 4°C). After post-fixation in 2% osmium tetroxide in Milloniq's phosphate buffer (pH 7.4, 1hr., 4°C) the tissue was dehydrated routinely in ethanol and embedded in Epon 812. The thin sections were stained with uranyl acetate in methanol and lead citrate then observed with either a Zeiss EM 9A or an Hitachi HS-8 electron microscope.

Ultrastructure of Granules Passing Through Frog Oocyte Nuclear Pores

1969 ◽  
Vol 27 ◽  
pp. 288-289
Author(s):  
Madison B. Cole
Keyword(s):  
Osmium Tetroxide ◽  
Rana Pipiens ◽  
Uranyl Acetate ◽  
Nuclear Pores ◽  
Thin Sections ◽  
Close Proximity ◽  
Perinuclear Cytoplasm ◽  
Frog Oocyte ◽  
Pass Through ◽  
Number Of Authors

Ovaries of Rana pipiens larvae were fixed in glutaraldehyde, post-fixed in osmium tetroxide, and embedded in epon. Thin sections of oocytes were routinely stained using methanolic uranyl acetate followed by lead citrate.The granule, designated M1, found in the primary oocytes is considered to pass through the nuclear pores by virtue of 1) its location in the nucleoplasm in close proximity to the nuclear envelope, 2) its contact with the nuclear pores, 3) its presence in aggregates in the perinuclear cytoplasm and A) its similarity to granules referred to by a number of authors (1-4) as material passing through nuclear pores.

Comparison of Three Common Fixatives on Bacteria

1977 ◽  
Vol 35 ◽  
pp. 348-349
Author(s):  
J.M. Hanson ◽  
R.M. Pfister ◽  
R.A. Smucker
Keyword(s):  
Bacillus Megaterium ◽  
Cell Walls ◽  
Uranyl Acetate ◽  
Ruthenium Red ◽  
Thin Sections ◽  
Internal Morphology ◽  
Cytoplasmic Membranes ◽  
Glutaraldehyde Solution ◽  
Background Material ◽  
Whole Mounts

The ultrastructure of the cytoplasmic membranes, cell walls, and mesosomes of Escherichia coli and Bacillus megaterium were found to be significantly affected by different fixation procedures.Logarithmic cells were divided into equal portions and different fixation procedures employed. A 6% glutaraldehyde solution followed by 1% 0s04 fixa-tion (1). The Ryter-Kellenberger (R-K) fixation (2) and Luft's ruthenium red (R-R) (3). The glutaraldehyde-OsO4 and R-K were post fixed with 2% and 0.5% uranyl acetate respectively. Thin sections were post stained with uranylacetate and lead citrate. One sub-portion of Bacillus megaterium was criti-cally-point dried. Glutaraldehyde-OsO4 critically-point dried whole mounts (Fig. 1) showed little background material and smooth walls. The R-K cells (Fig. 2) had irregular outer walls. The R-R cells (Fig. 3) were irregular and revealed external polysaccharide. There is a distinct difference in the internal morphology of the wall layers using the E. coli (Figs. 4,5,6). A heavier staining of the CM was observed in B. megaterium (Fig. 7).

Electron Microscopy as an aid to diagnosis in pediatric hematology/oncology

1989 ◽  
Vol 47 ◽  
pp. 1062-1063
Author(s):  
K. L. Saving ◽  
R. C. Caughey
Keyword(s):  
Electron Microscopy ◽  
Phosphate Buffer ◽  
Osmium Tetroxide ◽  
Uranyl Acetate ◽  
En Bloc ◽  
Thin Sections ◽  
Megakaryoblastic Leukemia ◽  
Pediatric Hematology ◽  
Transmission Electron ◽  
Microscopy Techniques

This presentation is designed to demonstrate how scanning and transmission electron microscopy techniques can be utilized to confirm or support a variety of unusual pediatric hematologic/oncologic disorders. Patients with the following diagnoses will be presented: (1) hereditary pyropoikilocytosis, (2) familial erythrophagocytic lymphohistiocytosis, (3) acute megakaryoblastic leukemia, and (4) pseudo-von Willebrand’s disease.All transmission and scanning electron microscopy samples were fixed in 2.5% glutaraldehyde, rinsed in Millonig’s phosphate buffer, and post-fixed with 1% osmium tetroxide. The transmission samples were then en bloc stained with 0.5% uranyl acetate, rinsed with Walpole ’ s non-phosphate buffer, dehydrated with graded series of ethanols and embedded with Epon 812 epoxy resin. Ultramicrotomy thin sections were stained with uranyl acetate and lead citrate and scanned using a JEOL-JEM 100C, The scanning samples were dehydrated with graded series of ethanols, critical point dried with CO2, gold-coated, and scanned using a JEOL-JSM 35. The peroxidase samples were fixed in 3% glutaraldehyde, incubated in diaminobenzidine (DAB), dehydrated with ethanol, embedded with Epon 812, and scanned without post-staining using a JEOL-JEM 100C.

Comparative study of ultrastructure of HIV and SIV

1990 ◽  
Vol 48 (3) ◽  
pp. 342-343
Author(s):  
Takashi Nakano ◽  
Shunro Imura ◽  
Masuyo Nakai
Keyword(s):  
Osmium Tetroxide ◽  
Staining Method ◽  
Culture Fluid ◽  
Ultrastructural Feature ◽  
Uranyl Acetate ◽  
Negative Staining ◽  
Thin Sections ◽  
Viral Particles ◽  
Infected Cells ◽  
Hiv 1

The ultrastructural feature of AIDS viruses (HIV and SIV) was observed by ultrathin-sectioning and negative staining method, and the surface structures of these viruses were compared.HIV-1 group including, HTLV-III and LAV-1 were used. And in HIV-2 group, LAV-2, which was isolated from a Senegalese AIDS patient, and GH-1, which was isolated from a Ghanan AIDS patient, were used. SIV group was used AGM-1, which was isolated from an African green monkey. The virions were used which was produced from Molt-4 cells after the infection of each viruses of these strains. The infected cells and viral particles, which were collected by centrifugation were fixed with 2% glutaraldehyde (GA), washed with PBS, and postfixed with 1% osmium tetroxide. The specimens were dehydrated in graded ethanol, and embedded in Epon 812. The thin sections were stained with uranyl acetate and lead citrate. On the other hand, for negative staining, 25% GA was added to the supernatant culture fluid and finally concentrated on 2% GA. Then virions were collected by ultracentrifugation.

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