Evaluation of alternative fixatives/protocols for the ultra-structural preservation of fast and slow growing mycobacteria

1996 ◽  
Vol 54 ◽  
pp. 814-815
Author(s):  
S. F Hayes ◽  
P. L. C. Small
Keyword(s):  
Tannic Acid ◽  
Freeze Substitution ◽  
Ruthenium Red ◽  
Human Pathogens ◽  
Mycobacterial Species ◽  
Pig Skin ◽  
Low Viscosity ◽  
Mycobacterium Aurum ◽  
Structural Preservation ◽  
Slow Growing

Various fixation formulas and protocols were examined to determine a routinely optimal fixation of Mycobacterial species in and out of tissues. These were evaluated by TEM and compared to results obtained using freeze substitution methods upon other Mycobacteria such as Mycobacterium aurum CIPT 1210005, M. fortuitum, M. phlei 425, M. kansasii and M. thermoresistible ATCC 19527Samples consisted of two slow growing mycobacterial species, both human pathogens; Mycobacterium tuberculosis, grown in M7H9 broth, and Mycobacterium marinum (1218 R & S variants), grown on M7H10 agar, with the latter also grown in tissue culture, and in guinea pig skin. Fixatives included: (1) glutaraldehyde/parafoimaldahyde/tannic acid in a phosphate/sucrose buffer, (2) paraformaldehyde/polylysine/periodate/glutaraldehyde (PLPG) in phosphate buffer followed by tannic acid and reduced osmium respectively, (3) PLP followed by tannic acid only without osmium, and (4) fixative 84-40 containing carbodiimide, glutaraldehyde and ruthenium red. Protocols varied in the length of time for fixation, types of buffers, solvents and in embedding schedules for Spurr's low viscosity resin.

Structural preservation and absolute contrast of catalase crystal sections prepared with tannic acid

1983 ◽  
Vol 41 ◽  
pp. 448-449
Author(s):  
C.W. Akey ◽  
M. Szalay ◽  
S.J. Edelstein
Keyword(s):  
Tannic Acid ◽  
Beef Heart ◽  
X Rays ◽  
Screw Axis ◽  
General Applicability ◽  
Thin Sections ◽  
Beef Liver ◽  
3 Dimensional ◽  
Dimensional Reconstruction ◽  
Structural Preservation

Three methods of obtaining 20 Å resolution in sectioned protein crystals have recently been described. They include tannic acid fixation, low temperature embedding and grid sectioning. To be useful for 3-dimensional reconstruction thin sections must possess suitable resolution, structural fidelity and a known contrast. Tannic acid fixation appears to satisfy the above criteria based on studies of crystals of Pseudomonas cytochrome oxidase, orthorhombic beef liver catalase and beef heart F1-ATPase. In order to develop methods with general applicability, we have concentrated our efforts on a trigonal modification of catalase which routinely demonstrated a resolution of 40 Å. The catalase system is particularly useful since a comparison with the structure recently solved with x-rays will permit evaluation of the accuracy of 3-D reconstructions of sectioned crystals.Initially, we re-evaluated the packing of trigonal catalase crystals studied by Longley. Images of the (001) plane are of particular interest since they give a projection down the 31-screw axis in space group P3121. Images obtained by the method of Longley or by tannic acid fixation are negatively contrasted since control experiments with orthorhombic catalase plates yield negatively stained specimens with conditions used for the larger trigonal crystals.

High-Resolution Scanning Electron Microscopy of Biological Specimens

1988 ◽  
Vol 46 ◽  
pp. 186-187
Author(s):  
M. Müller ◽  
R. Hermann
Keyword(s):  
High Resolution ◽  
Freeze Drying ◽  
Room Temperature ◽  
Structural Information ◽  
Freeze Substitution ◽  
Critical Point Drying ◽  
Sem Study ◽  
Major Factors ◽  
Structural Preservation ◽  
Preparation Techniques

Three major factors must be concomitantly assessed in order to extract relevant structural information from the surface of biological material at high resolution (2-3nm).Procedures based on chemical fixation and dehydration in graded solvent series seem inappropriate when aiming for TEM-like resolution. Cells inevitably shrink up to 30-70% of their initial volume during gehydration; important surface components e.g. glycoproteins may be lost. These problems may be circumvented by preparation techniques based on cryofixation. Freezedrying and freeze-substitution followed by critical point drying yields improved structural preservation in TEM. An appropriate preservation of dimensional integrity may be achieved by freeze-drying at - 85° C. The sample shrinks and may partially collapse as it is warmed to room temperature for subsequent SEM study. Observations at low temperatures are therefore a necessary prerequisite for high fidelity SEM. Compromises however have been unavoidable up until now. Aldehyde prefixation is frequently needed prior to freeze drying, rendering the sample resistant to treatment with distilled water.

Freeze-substitution of rye grass and ragweed pollen grains

1990 ◽  
Vol 48 (3) ◽  
pp. 686-687
Author(s):  
Liza B. Martinez ◽  
Susan M. Wick
Keyword(s):  
Cell Function ◽  
Pollen Grains ◽  
Freeze Substitution ◽  
Cell Types ◽  
Rapid Freezing ◽  
Rye Grass ◽  
Acrylic Resins ◽  
Allergenic Proteins ◽  
Cold Embedding ◽  
Structural Preservation

Rapid freezing and freeze-substitution have been employed as alternatives to chemical fixation because of the improved structural preservation obtained in various cell types. This has been attributed to biomolecular immobilization derived from the extremely rapid arrest of cell function. These methods allow the elimination of conventionally used fixatives, which may have denaturing or “masking” effects on proteins. Thus, this makes them ideal techniques for immunocytochemistry, in which preservation of both ultrastructure and antigenicity are important. These procedures are also compatible with cold embedding acrylic resins which are known to increase sensitivity in immunolabelling.This study reveals how rapid freezing and freeze-substitution may prove to be useful in the study of the mobile allergenic proteins of rye grass and ragweed. Most studies have relied on the use of osmium tetroxide to achieve the necessary ultrastructural detail in pollen whereas those that omitted it have had to contend with poor overall preservation.

scholarly journals Extracellular DNA of slow growers of mycobacteria and its contribution to biofilm formation and drug tolerance

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Aleksandr Ilinov ◽  
Akihito Nishiyama ◽  
Hiroki Namba ◽  
Yukari Fukushima ◽  
Hayato Takihara ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Drug Tolerance ◽  
Extracellular Dna ◽  
Exponential Growth Phase ◽  
Human Pathogens ◽  
Pellicle Formation ◽  
Phage Dna ◽  
Physiological Impact ◽  
Slow Growing ◽  
Growth And Reproduction

AbstractDNA is basically an intracellular molecule that stores genetic information and carries instructions for growth and reproduction in all cellular organisms. However, in some bacteria, DNA has additional roles outside the cells as extracellular DNA (eDNA), which is an essential component of biofilm formation and hence antibiotic tolerance. Mycobacteria include life-threating human pathogens, most of which are slow growers. However, little is known about the nature of pathogenic mycobacteria’s eDNA. Here we found that eDNA is present in slow-growing mycobacterial pathogens, such as Mycobacterium tuberculosis, M. intracellulare, and M. avium at exponential growth phase. In contrast, eDNA is little in all tested rapid-growing mycobacteria. The physiological impact of disrupted eDNA on slow-growing mycobacteria include reduced pellicle formation, floating biofilm, and enhanced susceptibility to isoniazid and amikacin. Isolation and sequencing of eDNA revealed that it is identical to the genomic DNA in M. tuberculosis and M. intracellulare. In contrast, accumulation of phage DNA in eDNA of M. avium, suggests that the DNA released differs among mycobacterial species. Our data show important functions of eDNA necessary for biofilm formation and drug tolerance in slow-growing mycobacteria.

Endogenous Opioids and Ruthenium Red Inhibit the Flare Reaction in the Pig Skin by Different Mechanisms

1991 ◽  
Vol 632 (1 Substance P a) ◽  
pp. 460-463 ◽  
Author(s):  
FR-K. PIERAU ◽  
R. ERNST ◽  
H. SANN ◽  
L. BARTHÓ
Keyword(s):  
Ruthenium Red ◽  
Endogenous Opioids ◽  
Pig Skin ◽  
Flare Reaction

Branching morphogenesis in the avian lung: electron microscopic studies using cationic dyes

Development ◽  
1986 ◽  
Vol 94 (1) ◽  
pp. 189-205
Author(s):  
Betty C. Gallagher
Keyword(s):  
Tannic Acid ◽  
Basal Lamina ◽  
Branching Morphogenesis ◽  
Ruthenium Red ◽  
Interparticle Spacing ◽  
Blue Staining ◽  
Mouse Lung ◽  
Alcian Blue Staining ◽  
Electron Microscopic ◽  
Avian Lung

The developing chick lung was examined in the electron microscope for intimate cell contacts between epithelium and mesenchyme, discontinuities in the basal lamina and substructure of the basement membrane. Cell filopodia were seen which crossed the basal lamina from both the epithelial and the mesenchymal cells. Ruthenium red and tannic acid staining of the basal lamina of the chick lung showed it to be thin and sometimes discontinuous at the tips compared to the more substantial basal lamina in the interbud areas. The bilaminar distribution of particles seen with ruthenium red is similar to those seen in the cornea and lens. With tannic acid staining, filaments could be seen which crossed the lamina lucida and connected with the lamina densa. Spikes perpendicular to the basal lamina were sometimes seen with a periodicity of approximately 110 nm. Alcian blue staining revealed structure similar to that seen by ruthenium red staining in the salivary and mammary glands, although the interparticle spacing was closer. Collagen was located in areas of morphogenetic stability, as has been seen by other investigators in different tissues. Collagen was coated with granules (probably proteoglycan) at periodic intervals when stained with ruthenium red. The fibrils were oriented circumferentially around the mesobronchus and were assumed to continue into the bud, but the fibres curve laterally at the middle of a bud. This orientation is opposite to that seen by another investigator in the mouse lung. In general, the observations made in the avian lung are similar to those seen in branching mammalian tissue. It is likely, therefore, that the chick lung uses strategies in its morphogenesis that are similar to those that have been elucidated previously in developing mammalian organs.

Histochemical and Ultrastructural study of the Platelet-Arterial Subendothelium Interaction

1979 ◽  
Author(s):  
Y.J. Legrand ◽  
J. Bariety ◽  
P. Birembaut ◽  
H. Michel ◽  
F. Fauvel ◽  
...  
Keyword(s):  
Tannic Acid ◽  
Platelet Adhesion ◽  
Ricinus Communis ◽  
Blood Perfusion ◽  
Rabbit Aorta ◽  
Human Platelets ◽  
Ruthenium Red ◽  
Ultrastructural Studies ◽  
Bacterial Collagenase ◽  
Before And After

In order to characterize the subendothelial macromolecules able to interact with platelets rabbit aortas were de-endothelialized and incubated either with chymotrypsin (24 h at 37° C), with a highly purified bacterial collagenase (2 h at 37° C), or with chymotrypsin followed by collagenase or the reverse. Histochemical ultrastructural studies were performed before and after blood perfusion, using three different staining procedures: tannic acid (for the microfibrillar structures and elastin), ruthenium red (for the polyanions) and two peroxidase-labelled lectins (concanavalin A and ricinus communis (for the detection of saccharidic determinants)). After chymotrypsin, the tannic acid +ve, ruthenium red +ve and lectin +ve microfibrils had been digested; platelet adherence to the remaining sparse collagen fibrils persisted. After collagenase, these microfibrils are preserved, collagen has been digested, but the platelets can still adhere. After combined incubation with both enzymes, elastin is the only remaining constituent and no platelet adhesion can be observed.These results show that in the rabbit aorta subendothelium besides collagen, microfibrillar glycoproteins are able to interact with human platelets.

scholarly journals The cell coat of the sensory and supporting cells of the rainbow trout saccular macula as demonstrated by reaction with ruthenium red and tannic acid.

1990 ◽  
Vol 38 (11) ◽  
pp. 1615-1623 ◽  
Author(s):  
K M Khan ◽  
J S Hatfield ◽  
D G Drescher
Keyword(s):  
Rainbow Trout ◽  
Tannic Acid ◽  
Ruthenium Red ◽  
Sensory Cells ◽  
Surface Coat ◽  
Basal Region ◽  
Apical Surface ◽  
Luminal Surface ◽  
Supporting Cells ◽  
Cell Coat

The surface of most cells is covered by glycoconjugates. The composition and thickness of the surface coat varies among different cell types. The purpose of the present study was to demonstrate the presence of and to characterize the cell coat surrounding the cells in the saccular macula of the rainbow trout. Tissues were fixed in Karnovsky's fixative containing either ruthenium red (0.5, 1, or 2%) or tannic acid (1, 2, or 4%). The apical surface of the sensory and supporting cells reacted with both agents. Varying the concentration of the compounds within a certain range did not significantly affect the degree of tissue staining. Whereas ruthenium red staining was distributed evenly along the luminal surface of the epithelium and along the length of the stereocilia, tannic acid formed electron-dense clumps on the luminal surface of sensory and non-sensory cells and in the basal region of the macular epithelium. The stereocilia of the sensory cells also exhibited tannic acid-positive, electrondense precipitate, particularly near the distal ends of these processes, while uniform staining of the plasma membrane was seen along their lengths. The results of this study suggest that the trout saccular macula is provided with extracellular microenvironments which may be necessary for functional integrity.

scholarly journals Evaluation of silicon and germanium retention in rat tissues and diatoms during cell and organelle preparation for electron probe microanalysis.

1975 ◽  
Vol 23 (5) ◽  
pp. 348-358 ◽  
Author(s):  
C W Mehard ◽  
B E Volcani
Keyword(s):  
Rat Liver ◽  
Freeze Substitution ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Rat Tissues ◽  
Cell Organelles ◽  
Thin Sections ◽  
X Ray ◽  
Low Viscosity ◽  
Silicon And Germanium

Chemical, radiochemical and x-ray microanalysis assays were used to define parameters of silicon (Si) retention during preparation og biologic samples (rat liver, spleen, kidney, lung, diatoms and cell organelles) for x-ray microanalysis, Due to its longer half-life 68-Fe was used in some cases to trace SI. Leaching of Si from cells and organelles by the aqueous preparation media was overcome by use of the freeze-substitution process. Cells were treated with 30% glycerol hypertonic sucrose medium to reduce ice damage. Embedment in Spurr's low viscosity epoxy resin medium caused no apparent Si loss. A semiquantitative evaluation showed 0.5 x 10-8 to 0.3 x 10-17 g detectable Si in isolated rat liver mitochondria in thin sections, which is within the instrument's range of detection. This study indicateds that the presence of Si in the mitochondria is not the rsult of contamination.

Visualization, adhesiveness, and cytochemistry of the extracellular matrix produced by urediniospore germ tubes of Puccinia sorghi

1991 ◽  
Vol 69 (9) ◽  
pp. 2044-2054 ◽  
Author(s):  
Rajendra Chaubal ◽  
V. A. Wilmot ◽  
Willard K. Wynn
Keyword(s):  
Electron Microscopy ◽  
Extracellular Matrix ◽  
Rust Fungus ◽  
Cetylpyridinium Chloride ◽  
Freeze Substitution ◽  
Ruthenium Red ◽  
Puccinia Sorghi ◽  
Temperature Scanning ◽  
Germ Tubes ◽  
Scanning Electron

Adherence of germinating urediniospores of the common maize rust fungus (Puccinia sorghi Schw.) to substrata was studied by ultrastructural and cytochemical examination of extracellular matrix produced by germ tubes in conjunction with measurements of adhesion to plastic and glass surfaces. Copious amounts of extracellular matrix on germ tubes could consistently be visualized by scanning and transmission electron microscopy only when (i) a cationic detergent (cetylpyridinium chloride, polydiallyldimethylammonium chloride) or a cationic stain (ruthenium red, alcian blue, cuprolinic blue) was added to the fixation solutions, (ii) germ tubes were fixed by rapid-freezing and freeze-substitution and observed with a scanning electron microscope, or when (iii) germ tubes were observed in a frozen-hydrated state by low-temperature scanning electron microscopy. Incubation of germinated spores with dilute alkalies (NaOH, KOH), pronase E (nonspecific protease), and laminarinase (β-1,3 (1,3; 1,4-glucanase) removed the extracellular matrix and detached germ tubes from surfaces. Treatments with water, dilute acids, ionic and neutral detergents, organic solvents, hydrocarbons, and several polysaccharide-degrading enzymes did not remove the extracellular matrix and also did not detach germ tubes. These results, together with staining patterns obtained with lectins and other polysaccharide-specific reagents, indicate that the extracellular matrix is composed mainly of glycoproteins rich in acidic amino acids and β-1,3-glucan polymers, and that it is probably responsible for the adhesion of the rust germ tubes to the host leaf surfaces. Key words: Puccinia sorghi, germ tube adhesion, extracellular matrix, cytochemistry.

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