Showing papers on "Glutaraldehyde published in 1972"

Theoretical and practical aspects of glutaraldehyde fixation.

Abstract: This review first considers the many structures put forward for glutaraldehyde, and the purification of the commercial material for chemical, histological and histochemical studies. Some practical and theoretical problems of tissue fixation with glutaraldehyde, including artefacts, are then discussed. The chemical reactions with amino acids and proteins are considered next together with the physical changes in the proteins during the reactions. The known reactions of glutaraldehyde with nucleic acids, lipids and mucosubstances are explored briefly.

Phosphotungstic Acid-Chromic Acid as a Selective Electron-Dense Stain for Plasma Membranes of Plant Cells

Abstract: A mixture consisting of 1% phosphotungstic acid (PTA) in 10% chromic acid (CrO3) selectively stains the plasma membrane of plant cells. Whole tissue or pelleted cell fractions are prepared for electron microscopy using conventional methods including glutaraldehyde fixation and OsO4 postfixation, dehydration in acetone and embedding in Epon. To stain the plasma membrane, thin sections are transferred with a plastic loop to the surface of a 1% aqueous solution of periodic acid for 30 min for destaining. Following transfer through 5 distilled water rinses, the sections are exposed to the PTA-CrO3 mixture for 5 min, rinsed and mounted on grids for viewing with the electron microscope. The selectivity of the stain is retained in homogenates and serves to identify the plant plasma membrane in cell fractions.

Fixation by means of glutaraldehyde-hydrogen peroxide reaction products

Abstract: CAMILLO PERACCHIA and BRANT S. MITTLER . From the Department of Anatomy, Duke University Medical Center, Durham, North Carolina 27706, and the Department of Physiology, University of Rochester, School of Medicine and Dentistry, Rochester, New York 14642 . Dr. Peracchia's present address is the Department of Physiology, University of Rochester, School of Medicine and Dentistry, Rochester, New York 14642 .

Physicochemical effects of aldehydes on the human erythrocyte.

Abstract: The effects of formaldehyde, acetaldehyde, and glutaraldehyde on human red blood cells were investigated. It was found that (a) The surface negative charge of the erythrocytes at pH 7 was increased 10% by glutaraldehyde, but not by the other two aldehydes. (b) The effect of incomplete fixation of the red blood cells was demonstrated by hemoglobin leakage studies The leakage of hemoglobin subsequent to formaldehyde treatment was especially pronounced Acetaldehyde-fixed cells showed some leakage of hemoglobin after an hour of exposure to the fixative, whereas glutaraldehyde-fixed cells showed no hemoglobin leakage. (c) All three aldehydes caused K+ leakage during fixation. The concentrations of K+ in the fixing solutions all reached the same level, but whereas the leakage with glutaraldehyde was immediate, that with formaldehyde was more gradual and that with acetaldehyde reached a steady state only after 24 hr. (d) The effects of the aldehydes on red cell deformability and swelling revealed that glutaraldehyde hardened the cells within 15 min, formaldehyde within 5 hr, while acetaldehyde required at least 24 hr to produce appreciable fixation. (e) The hematocrit changes accompanying the fixation process depended upon cell volume changes and loss of deformability.

The demonstration of acid phosphatase in in vitro cultured tissue cells. Studies on the significance of fixation, tonicity and permeability.

Abstract: 1. Methods for the fine structural demonstration of acid phosphatase were studied in monolayers of in vitro cultured cells after fixation with glutaraldehyde. 2. Inactivation of enzyme activity occurred rapidly during the initial phase of glutaraldehyde fixation. 3. Fixation for more than 5 min did not cause further marked inactivation of enzyme activity. 4. Stabilization of the cells for cytochemical incubations required a fixation for at least 30 min in glutaraldehyde. 5. The total osmolality of the fixative was of minor importance, in contrast to the major importance of effective osmolality, for obtaining optimum cytochemical and ultra-structural results. 6. Following proper fixation, the osmotic strength of the washing and incubation solutions was not critical. 7. With short fixation times, the composition of the washing and incubation solutions was of major importance. 8. Dimethyl sulphoxide in washing and incubation media was effective in shortening incubation times (thereby preventing the occurrence of unspecific precipitates and derangement of fine structure).

I. A study of passive potassium efflux from human red blood cells using ion specific electrodes. II. Quantitation of human red blood cell fixation by glutaraldehyde

Abstract: I. A STUDY OF PASSIVE POTASSIUM EFFLUX FROM HUMAN RED BLOOD CELLS USING ION SPECIFIC ELECTRODES It is shown that, by using ion specific electrodes, the small potassium leakage induced by ouabain in human erythrocytes can be measured continuously and precisely. Upon addition of isotonic sucrose solution to a suspension of red cells in physiological saline the passive (ouabain-insensitive) potassium efflux is directly proportional to the chloride ratio. (i. e., an exponential function of the total membrane potential). The same result is obtained upon addition of hypertonic sucrose solution, suggesting that neither osmolarity nor intracellular concentrations have any influence on the passive potassium efflux. The independence of the potassium efflux and osmolarity can be verified by addition of glucose to the cell suspension. Glucose penetrates the red cells leaving the intracellular volume,and thus the membrane potential as well as the intracellular concentrations, unchanged. Adding water or hypertonic sodium chloride solution to red blood cell suspensions shows that the potassium efflux increases slightly in more concentrated salt solutions. Inasmuch as this effect can be interpreted as a pure ionic strength effect, the experiments corroborate the hypertonic sucrose solution experiments in demonstrating no dependence of the potassium efflux upon intracellular concentrations. The results of this investigation as well as other studies (LaCelle and Rothstein 1966, Donlon and Rothstein 1969, Cotterrell and Whittam 1971) show that the passive permeability of the human red blood cell to potassium depends uniquely on the membrane potential near physiological conditions, while it depends on parameters such as pH or concentrations for values of the membrane potential over 40mV. This suggests that two different mechanisms of transport might be involved: one would control the permeability under normal conditions; the other would represent a leak through the route normally used by anions and become important only under extreme conditions. II. QUANTITATION OF HUMAN RED BLOOD CELL FIXATION BY GLUTARALDEHYDE The uptake of glutaraldehyde by human red blood cells has been measured as a function of time by a freezing point osmometer. The rate of attachment of glutaraldehyde to the cell proteins is high over the first hour, declining to zero over a period of a few days. The number of glutaraldehyde molecules cross-linking with each hemoglobin molecule is of the order of 200, in reasonable agreement with the calculated number of attachment sites. The cell membrane is immediately highly permeable to glutaraldehyde. Selective permeability to ions is lost during fixation. Ionic equilibrium is obtained only after a few hours. An optimum fixation technique for shape preservation is suggested.

Glutaraldehyde--its purity and stability.

Abstract: Six commercially available glutaraldehydes were tested spectrophotometrically for their purity. These results were compared with glutaraldehyde purified by two experimental techniques. Ten sets of storage conditions were chosen and aliquots of purified glutaraldehyde were stored under these conditions for eight months. These samples enabled the reappearance of the main contaminant, absorbing strongly at 235 nm, to be examined with regard to temperature, light and the availability of oxygen. From the results the optimum criteria for storage of pure glutaraldehyde were elucidated. The results lend weight to the view that the main contaminent in stored glutaraldehyde is a polymer of glutaraldehyde and not glutaric acid.

Osmolarity of osmium tetroxide and glutaraldehyde fixatives.

Abstract: The evidence available to date for the importance of fixative osmolarity is considered together with some observations on the volume changes of crab axons after fixation by osmium tetroxide and glutaraldehyde. The results obtained are compared with those obtained from crab axons and from amphioxus skin cells which had been processed and examined with the electron microscope after initial fixation in fixatives of different composition. It is concluded that the osmolarity of the fixative vehicle is of considerable importance when the fixing agent is glutaraldehyde but is of less importance when the fixing agent is osmium tetroxide or a mixture of the two agents. Preliminary observations upon crab axons fixed with glutaraldehyde in a vehicle approximating to the internal composition of the cells suggest that this approach to the design of fixative vehicles may be useful.

Contact dermatitis from glutaraldehyde.

Abstract: Allergic contact dermatitis can result from occasional or incidental contact with glutaraldehyde used for sterilization of medical and dental equipment, as well as from topical application of the compound in dermatologic treatment. The patients described had positive patch tests to glutaraldehyde and glutaraldehyde-tanned leather. It remains to be shown whether glutaraldehyde in leather can induce sensitization.

Inhibition of Sindbis Virus Release by Media of Low Ionic Strength: an Electron Microscope Study

Abstract: Release of Sindbis virus from infected cells is inhibited by lowering the ionic strength of the medium. To determine the nature of the inhibited step, we examined, by electron microscopy, both freeze-etched and thin-sectioned preparations which had been fixed with either glutaraldehyde or formaldehyde. Inhibitory medium had two different effects on Sindbis virus release: virus budding was partially inhibited, and those virions which did mature were precipitated on the surface of the cell. Freeze-etched, inhibited cells showed very few viral buds. After shift to normal medium, the number of budding virions increased dramatically, far exceeding the quantity found in normal controls. Thus, low ionic strength medium clearly inhibited an early stage of virus maturation. The results were the same regardless of the fixative. Thin sections of glutaraldehyde-fixed, inhibited cells contained large extracellular aggregates of mature virus which were not present in similar, formaldehyde-fixed preparations. Fixation of radioactively-labeled, inhibited cultures revealed that approximately half of the virus that could be released from inhibited cells by raising the ionic strength of the medium could also be released by formaldehyde, but not by glutaraldehyde. This fraction probably represents mature virus attached to the cell surface by the ionic conditions.

Filamentous and matrix components of skeletal muscle Z-disks.

Abstract: The fine structural appearance of Z-disk lattices in vertebrate skeletal “fast” muscle varies depending upon whether osmium or glutaraldehyde has been employed as the primary fixative. Prior investigators have attributed the differences to change in the extent of actin filament overlap within the Z-disk and/or to rearrangement of Z-disk filaments. Adult frog and young newt “fast” muscle has been studied under various degrees of stretch, with several different aldehyde and osmium fixation procedures, and after plastic section digestion techniques utilizing Pronase or pepsin. Serial cross sections of Z-disks were correlated with oriented cross and longitudinal sections. Fixation with collidine-buffered osmium and veronal acetate-buffered glutaraldehyde seems to provide the greatest and most distinctly contrasting differences. A consistently arranged phase, the filamentous lattice, can be discerned after either fixation. However, a second phase, termed “Z-disk matrix,” appears variable, perhaps due to extraction during primary osmium fixation procedures. Glutaraldehyde-fixed frog muscle Z-disks display a copious matrix, one which is seldom totally depleted by osmium fixation. In young newt muscle Z-disks, little matrix is present after glutaraldehyde fixation and none of it remains after primary osmium. In Z-disks fixed by either method, matrix that is retained appears to be deposited in lattice-like patterns. It is suggested that these matrix patterns, or their loss, are the basis for the varying images of Z-disks observed under diferent fixation conditions and that the filamentous lattice is relatively stable. The Z-disk is more rapidly obliterated by Pronase or pepsin digestion than is any other muscle component, including actin (which appears notably unreactive). The rapid digestion effect is limited to the region postulated to include the matrix phase. Models for the structural interrelationship of filamentous and matrix phases are discussed and compared to prior Z-disk models.

Osmolar concentration and fixation of mycoplasmas.

Abstract: Broth cultures of Acholeplasma laidlawii were fixed with various concentrations of cacodylate-buffered glutaraldehyde. The shape and ultrastructure of the organisms varied with the osmolar concentration of the fixative. When the fixation mixture was hypertonic to the culture medium, ultrathin sections suggested that the cells had shrunk. Phosphate buffer, sodium chloride, or sucrose at comparable osmolaities had the same effect as sodium cacodylate. Glutaraldehyde itself also contributed to the osmotic effects of the fixation mixture but to a lesser extent than salts or sucrose, to which the cell membrane is impermeable. The osmolar concentration of the fixation mixture seemed of greater importance than pH in determining morphology. The mycoplasma was still susceptible to damage by high concentrations of cacodylate after fixation with 2.5% glutaraldehyde. The best procedure was to fix and wash the organism under conditions isotonic with the growth medium. These conditions were also satisfactory for a filamentous mycoplasma, Mycoplasma orale.

Fixation of nitrogenous materials

Abstract: Dialdehydes, e.g., glyoxal or glutaraldehyde are used to fix nitrogenous materials, e.g., amino acids, proteins, enzymes, antigens, hormones, etc., to insoluble supports which form gels in aqueous media, e.g., polyacrylamide or polysaccharides such as agar-agar.

Effect of Glutaraldehyde on the Outer Layers of Escherichia coli

Abstract: Summary: Sodium lauryl sulphate (SLS) at pH 3 and 8 lysed cell walls of Escherichia coli. Pretreatment with glutaraldehyde at pH 3 and at pH 8 prevented this lysis. SLS induced maximum lysis of E. coli cells at 40°; pretreatment of cells with glutaraldehyde prevented this lysis also. Electrophoretic studies indicated that glutaraldehyde accumu lated on the surface of E. coli cells more rapidly in acid than in alkaline conditions, and that it blocked amino groups on the surface layer of Bacillus subtilis spores. The relationship of these findings to the bactericidal efficiency of glutaraldehyde in acid and alkaline solution is discussed.

Nutritive value of formaldehyde-treated rapeseed meal for dairy calves

Abstract: Treatment of oilseed meals with formaldehyde (FA) and glutaraldehyde (GA) significantly (P 0.05) in dry matter consumption, daily gain, or feed efficiency was observed. Total volatile fatty acids (VFA) (mmoles/100 ml) concentration was significantly higher (P 0.05) between the two groups. In the N balance and dige...

The hydration and polymerisation of succinaldehyde, glutaraldehyde, and adipaldehyde

Abstract: The 1H n.m.r. spectra of solutions of succinaldehyde, glutaraldehyde, and adipaldehyde in deuterium oxide have been studied. At room temperature, the major component of solutions of succinaldehyde and glutaraldehyde is a cyclic monohydrate which is accompanied by the free aldehyde OCH·CH2·[CH2]n·CH2CHO, the acyclic monohydrate (DO)2CH·CH2·[CH2]n·CH2·CHO, and the dihydrate (DO)2CH·CH2·[CH2]n·CH2·CH(OD)2; at higher temperatures the free aldehyde content increases, mainly at the expense of the cyclic monohydrate. Adipaldehyde, by contrast, forms only the acyclic dihydrate. Although the undiluted aldehydes polymerise readily, their neutral aqueous solutions are stable for Iong periods; acidification induces rapid polymerisation. A common mechanism is suggested for hydration and polymerisation.

Synthesis of δ‐Lactones from Glutaraldehyde

Abstract: A novel and general synthesis of δ-lactones from glutaraldehyde is described. The dialdehyde is first reacted with an alkyl or substituted alkyl Grignard reagent to afford a δ-hydroxyaldehyde in good yield. These aldehydes exist preferentially in the cyclic hemiacetal form (δ-lactols). Oxidation of the latter compounds to give δ-lactones is readily achieved with silver oxide or bromine. The δ-lactols and δ-lactones serve as intermediates for the total synthesis of steroids.

Improved preservation of alkaline phosphatase in salivary glands of the cat

Abstract: The effects of tissue preparation on the localization of alkaline phosphatase were assessed at the light and electron microscopical levels. Glutaraldehyde-containing solutions were more inhibitory than formaldehyde alone but appeared to give betterin situ immobilization of the enzyme. The inhibitory effects of glutaraldehyde solutions were related especially to temperature and time and not necessarily to material absorbing at 235 nm. Distilled glutaraldehyde was the only form of glutaraldehyde to give consistently good results with least inhibition. Snap-freezing of pre-fixed tissue, after washing in a sucrose-containing solution, gave satisfactory results without undue ice-crystal formation. Immersion in dimethylsulphoxide before snap-freezing gave less good localization with greater diffusion of reaction product. Use of a Sorvall tissue-chopper on unfrozen tissue did not produce satisfactory sections. Free-floating sections of pre-fixed material appeared less inhibited than glass-mounted sections. The most satisfactory results were obtained after per-arterial perfusion with a 1% distilled glutaraldehyde-0.8% formaldehyde mixture, containing dextran, for up to 5 min followed by formaldehyde-dextran. The results were uniform; there was stronger staining of stromal surfaces of myoepithelial cells than previously, basal duct cells were also stained and occasional staining between acinar cells was now evident for the first time.

Control of Cross-Contamination

Abstract: High-speed, air-driven handpieces and dental burs are contaminated after use and thus they can be a source of cross-contamination of patients. The effectiveness of the disinfectant solutions of benzalkonium chloride and aqueous activated 2% glutaraldehyde was compared in tests involving suspensions of two microorganisms. The glutaraldehyde solution was bacteriocidal within five minutes in tests involving Streptococcus pyogenes, and within three hours in tests with Bacillus stearothermophilus. Benzalkonium chloride appeared to be less effective.