Showing papers on "Glutaraldehyde published in 1980"

Design of an artificial skin. II. Control of chemical composition.

Abstract: Detailed methodology is described for the reproducible preparation of collagen--glycosaminoglycan (GAG) membranes with known chemical composition. These membranes have been used to cover satisfactorily large experimental full-thickness skin wounds in guinea pigs over the past few years. Such membranes have effectively protected these wounds from infection and fluid loss for over 25 days without rejection and without requiring change or other invasive manipulation. When appropriately designed for the purpose, the membranes have also strongly retarded wound contraction and have become replaced by newly synthesized, stable connective tissue. In our work, purified, fully native collagen from two mammalian sources is precipitated from acid dispersion by addition of chondroitin 6-sulfate. The relative amount of GAG in the coprecipitate varies with the amount of GAG added and with the pH. Since coprecipitated GAG is generally eluted from collagen fibers by physiological fluids, control of the chemical composition of membranes is arrived at by crosslinking the collagen--GAG ionic complex with glutaraldehyde, or, alternately, by use of high-temperature vacuum dehydration. Appropriate use of the crosslinking treatment allows separate study of changes in membrane composition due to elution of GAG by extracellular fluid in animal studies from changes in composition due to enzymatic degradation of the grafted or implanted membrane in these studies. Exhaustive in vitro elution studies extending up to 20 days showed that these crosslinking treatments insolubilize in an apparently permanent manner a fraction of the ionically complexed GAG, although it could not be directly confirmed that glutaraldehyde treatment covalently crosslinks GAG to collagen. By contrast, the available evidence suggests strongly that high-temperature vacuum dehydration leads to formation of chemical bonds between collagen and GAG. Procedures are described for control of insolubilized and "free" GAG in these membranes as well as for control of the molecular weight between crosslinks (Mc). The insolubilized GAG can be controlled in the range 0.5--10 wt. % while "free" GAG can be independently controlled up to at least 25 wt. %; Mc can be controlled in the range 2500--40,000. Studies by infrared spectroscopy have shown that treatment of collagen--GAG membranes by glutaraldehyde or under high-temperature vacuum does not alter the configuration of the collagen triple helix in the membranes. Neither do these treatments modify the native banding pattern of collagen as viewed by electron microscopy. Collagen--GAG membranes appear to be useful as chemically well-characterized, solid macromolecular probes of biomaterial--tissue interactions.

Biological effects of residual glutaraldehyde in glutaraldehyde‐tanned collagen biomaterials

Abstract: Glutaraldehyde is commonly used to control physical and biological properties of collagen structure by means of intramolecular and/or intermolecular crosslinking of collagen molecules. Solubility, antigenicity, and biodegradation of naturally occurring or reconstituted collagenous matrices are effectively reduced by glutaraldehyde treatment. Adverse biological reactions to glutaraldehyde have been limited to infrequent contact dermatitis and to biocidal effects which are exploited in chemical sterilization media. In the present study of glutaraldehyde-tanned collagen sponge, the presence of glutaraldehyde was correlated with cytotoxic effects upon fibroblasts in tissue culture and foreign body giant cell reaction to bioimplants of the sponge. Fibroblast growth in tissue culture is 99% inhibited at media concentrations of 3.0 ppm glutaraldehyde. Extracts of glutaraldehyde collagen sponge in aqueous media at pH 7 and 4.5 yielded 6 micrograms and 65 micrograms glutaraldehyde per gram of collagen sponge, respectively. The yield increased tenfold at pH 4.5. Observations indicate that leaching of the glutaraldehyde from glutaraldehyde-tanned collagen sponge is sufficient to produce potentially adverse cellular effects both in vivo and in vitro.

Use of glutaraldehyde as a coupling agent for proteins and peptides.

Abstract: Publisher Summary This chapter discusses the use of glutaraldehyde as a coupling agent for proteins and peptides. In immunochemical study, the need arises to link proteins to particles, to polymerize proteins, or to form covalent conjugates of proteins and smaller peptides. The availability of reagents that are simple and effective, while largely preserving the native antigenicity of the proteins and peptides under study, is of considerable utility. Glutaraldehyde is such a reagent and has been successfully used for all the above applications. Glutaraldehyde has been employed for coupling adenocorticotropic hormone (ACTH) and glucagon to larger proteins. The resulting conjugates were uniformly antigenic in rabbits. There have also been attempts to identify the reaction products of glutaraldehyde with the model compounds 6-aminohexanoic acid and α-N-acetyllysine. The ultraviolet, infrared, and nuclear magnetic resonance (NMR) spectra of a purified product, from the reaction of glutaraldehyde, with 6-aminohexanoic acid, has led to postulate those compounds of the type that have a polymeric quaternary pyridinium structure. Basics of the reactions of glutaraldehyde with proteins are discussed in this chapter. Immunogenicity of cytochrome c polymers in rabbits is described. Protein–particle conjugation is also discussed in this chapter.

Use of Aldehyde Fixatives to Determine the Rate of Synaptic Transmitter Release

Abstract: Aldehyde fixation continues to be useful to prepare synapses for freeze-fracture, but it may increase the rate of transmitter release. The effects of different aldehyde fixatives on spontaneous quantal release (m.e.p.p.s), and on the corresponding synaptic vesicle exocytosis at frog nerve-muscle synapses were investigated with the hope of finding a way to minimize side effects of fixation. Increases in m.e.p.p.s of up to 50 S-1 occurred during fixation, despite the species of aldehyde used in the fixative, and this fixative effect decreased only slightly as aldehyde concentration was increased. Increases in m.e.p.p frequency were not blocked by tetrodotoxin, by lowering external calcium and raising external magnesium concentration, or by lowering the total osmotic strength of the fixative. The smallest increase in m.e.p.p. frequency was in 3% glutaraldehyde and corresponded to the lowest level of synaptic vesicle exocytosis seen by freeze-fracture, 0.15 per micrometers of active zone. The effects of aldehyde fixation on m.e.p.p. frequency and synaptic vesicle exocytosis could not be avoided, but this study suggests how its effect on morphological changes in synapses might be minimized.

Synthesis and characterization of poly/glutaraldehyde/ - A potential reagent for protein immobilization and cell separation

Abstract: The aldol condensation of aqueous glutaraldehyde in the pH range 7-13.5 yielded water-soluble and water-insoluble poly(g1utaraldehyde) (PGL), the molecular weight of which was of the order of 12 to 20000. The structure of PGL was elucidated by means of UV, IR spectrophotometry, electrochemical studies, and the analysis of reaction products of PGL with hydroxylamine hydrochloride. The structure of PGL polymers prepared in a wide pH range was found to be similar. The main differences consisted of changes in the concentration of functional groups. Sufficient evidence was obtained to explain the presence in PGL of the primary hydroxyl and of the carboxyl groups as due to a Cannizzaro reaction. Electrochemical studies confirmed the spectrophotometric evidence of the presence of conjugated aldehyde groups. Recent investigations of water-soluble or -insoluble PGL or of PGL in the form of microspheres indicate that these polymers may yield important immunoreagents for biological research. Glutaraldehyde has many applications in different areas. It is used as a protein cross-linking agent,' for fixation of living cells2 or t i ~ s u e s , ~ and for sterilization of hospital e q ~ i p m e n t ; ~ it was also found to serve as an efficient binding agent of antibodies to micro sphere^.^ Glutaraldehyde was used in a variety of reactions for relatively long periods of time at physiological pH. It was only recently realized that under these conditions a considerable amount of glutaraldehyde is polymerized through the aldol condensation mechanism,6 and many reported reactions of glutaraldehyde are actually reactions of glutaraldehyde as well as of poly(g1utaraldehyde). Although a large number of studies were carried out in the past on the nature of aqueous gl~taraldehyde,~-'' little information is available on the structure of the solid aldol condensation product which was frequently considered to be an impurity associated with glutaraldehyde.12 The present study showed that polymerization of glutaraldehyde under basic conditions results in the production of water-soluble and -insoluble polymers and that these polymers contain nonconjugated aldehyde, conjugated aldehyde, hydroxyl, and carboxyl groups. The weight ratio of the soluble to the insoluble polymer and the concentration of the functional groups were found to be dependent on the pH of the polymerization medium. Poly(glutara1dehyde) might be used as a new reagent in protein chemistry and other areas. Only recently the polymer was found to constitute a valuable new reagent for the immobilization of antibodies on solid substrate^.'^ Furthermore, a new method was developed for the preparation of poly(glutara1dehyde) in the form of micro~pheres . '~ The microspheres were used for cell labeling and cell separation and are suitable also for immobilization of enzymes, drugs, and proteins. Experimental Section (a) Reagents. The following materials were acquired from commercial sources and were used without any further treatment: pyridine, phthalic anhydride, and hydroxylamine hydrochloride (Matheson Coleman and Bell), fluorescein isothiocyanate (Polysciences), tetraethylammonium perchlorate (Southwestern Analytical Co.), and ferrofluid (Ferrofluidic Co., Burlington, Mass.). Aqueous glutaraldehyde (Aldrich) was purified by treatment with activated carbon followed by filtration. Dimethylformamide, (DMF), dimethyl sulfoxide (Me2SO) and crotonaldehyde were vacuum distilled. (b) Apparatus. Infrared and UV spectra were obtained with a Fourier transform IR (FTS-l5C, Houston Instruments) and a Cary 219 spectrophotometer (Varian), respectively. Gel permeation chromatography (GPC) was carried out with the high-pressure liquid chromatography Model 6000, fitted with a refractive index detector (Water Associates). The GPC was carried out with dimethylformamide as a solvent and a column of styragel lo5, lo4, lo3 8, (pore size); polystyrene was used for calibration. Dupont thermal analyzer 900, Model 950 TGA, was used for thermogravimetric analysis. Polarograms were obtained with a Princeton Applied Research (PAR) Model 174 polarographic analyzer and recorded with a Hewlett Packard Model 7004 x-y recorder. Cyclic voltammograms were obtained by means of a PAR Model 173 potentiostat driven by a conventional signal generator. The voltammograms were recorded with the x-y recorder. Controlled potential coulometry was studied with the PAR Model 173 potentiostat equipped with a Model 179 digital coulometer. The reference calomel electrode was isolated from the main cell compartment by means of a fritted glass disk. The auxiliary electrode was a platinum wire (diameter 0.076 cm) sealed in soft glass. The working electrode was a hanging mercury drop electrode. (c) Synthes is of PGL. Glutaraldehyde was added to an appropriate deaerated buffer solution and then the mixture was placed on a mechanical shaker for 72 h. The white-yellowish precipitated polymer was filtered, washed with water, and then dried under vacuum at 45 \"C. The mother liquor was dialyzed extensively against distilled water and lyophilized in order to obtain the soluble PGL in solid form. The synthesis of PGL microspheres was already described.14 (d) Aldehyde Group Determination. The aldehyde content of PGL was determined from the percent nitrogen of the oxime prepared by the heterogeneous reaction of PGL with aqueous hydroxylamine hydr~chlor ide. '~~ '~ PGL (50 mg) was shaken a t room temperature for 24 h with 500 mg of hydroxylamine hydrochloride. The polymer was then filtered, washed with water, and dried under vacuum a t 45 \"C. A similar nitrogen content was obtained when the reaction was carried out a t 60 \"C or a t room temperature but a t pH 6.0. (e) Carboxy1,Group Determination. The carboxyl content of the polymer in the salt form was determined by ashing the samples and in the acid form by titration of the polymer dissolved in DMF/H20 (1:l) with 0.3 M NaOH. Similar results were obtained when the titration was carried out in warm pyridine. The agreement between the ashing method and the titration method was of the order of 15%. (f) Hydroxyl Group Determination. The determination of the hydroxyl content was based on the phthalation method.17-19

Factors determining degree of inflation in intratracheally fixed rat lungs

Abstract: The total lung capacity (TLC) of rats was measured in vivo and was compared to the displacement volume of the lungs following intratracheal fixation with glutaraldehyde or formaldehyde solution. When glutaraldehyde was used the speed of infusion of the fixative was an important factor in the final degree of lung inflation achieved. With a low rate of fixative infusion and a final pressure of 20 cm of fixative the glutaraldehyde-fixed lungs inflated to 55% TLC. With a high initial flow of glutaraldehyde and a final pressure of 20 cm of fixative the lungs inflated to 84% TLC. Fixation of lungs inside the intact chest wall was found to result in a higher degree of inflation. With a reservoir height of 20 cm and a low rate of fixative infusion lungs fixed in situ reached 74% TLC, whereas lungs fixed in situ, but from animals that have been exsanguinated prior to fixation, inflated to only 58% TLC. This suggests that the volume of the blood in the lungs prior to infusion of glutaraldehyde influences the degree of inflation achieved. Formaldehyde-fixed lungs required 72 h to be completely fixed and they were inflated to 90% TLC when a reservoir height of 20 cm was used. Because of the slow rate of fixation using with formaldehyde solution the rate of infusion was found not to limit the degree of inflation that could be achieved.

Effect of aldehyde cross-linking on human dermal collagen implants in the rat.

Abstract: The fate of s.c. implants of fibrous, trypsin-purified human dermal collagen and collagen cross-linked with formaldehyde and glutaraldehyde has been studied in rats. Dermal collagen, and untreated skin implants, underwent resorption associated with a pronounced round-cell reaction. While collagen implants cross-linked with solutions of 0.04% and 0.08% formaldehyde became reduced in size, those cross-linked with 1% formaldehyde and 0.01%, 0.02% and 0.04% glutaraldehyde, although undergoing some collagen remodelling, retained their original size over the 25-week period of study. At 5 weeks the aldehyde cross-linked implants showed their greatest cellularity, reaching a lower, more stable cell population by 18 weeks. More round cells were seen at 5 weeks, particularly after formaldehyde cross-linking, than a later times when few were present. The results indicate that aldehyde-stabilized preparations of heterograft dermal collagen could have applications in the repair of tissue defects in man.

Glutathione production coupled with an ATP regeneration system

Abstract: Escherichia coli cells possessing glutathione synthetase and acetate kinase activities were immobilized with carrageenan gel. To enhance the operational stability, immobilized cells were treated with hardening agent, glutaraldehyde in the presence of hexamethylenediamine. The continuous production of glutathione was investigated using the column packed with immobilized Escherichia coli cell preparations. Glutathione was continuously produced by this column in the presence of acetyl phosphate and the half-life of this column was calculated to be 8 days at the flow rate of S.V.=0.1 h−1 at 37°C.

Shrinkage of lung after chemical fixation for analysis of pulmonary structure-function relations.

Abstract: Glutaraldehyde is widely used to chemically fix lungs for analysis of pulmonary structure-function relations. Accurate interpretation of observations on fixed tissue requires a clear definition of any artifacts, such as tissue shrinkage, resulting from fixation with glutaraldehyde. Two experimental procedures were used in this study to examine possible shrinkage artifacts resulting from fixation of lung by glutaraldehyde. In the first, isolated perfused dog lungs were rapidly frozen at different transpulmonary pressures. Samples were then freeze substituted at -50 degrees C using 70% ethylene glycol with and without fixatives present. In the second series of experiments, the left lungs of mongrel dogs were fixed by vascular perfusion with glutaraldehyde at different transpulmonary pressures. In both series of experiments any changes in linear dimensions resulting from the fixation procedure were measured. Also, the presence of aldehyde was demonstrated by a positive reaction with Schiff reagent. The results demonstrate that lung tissue fixed either by vascular perfusion or freeze substitution tends to shrink to about the same extent. This shrinkage is reasonably constant at about 9% for transpulmonary pressures of 5 and 15 cmH2O and increases to about 15% when the transpulmonary pressure reaches 25 cmH20.

In-situ cross linking of polyvinyl alcohol

Abstract: A method of producing a cross-linked polyvinyl alcohol structure, such as a battery separator membrane or electrode envelope. An aqueous solution of a film-forming polyvinyl alcohol is admixed with an aldehyde cross-linking agent at basic pH to inhibit cross-linking. The cross-linking agent, preferably a dialdehyde such as glutaraldehyde, is used in an amount of from about 1/2 to about 20% of the theoretical amount required to cross-link all of the hydroxyl groups of the polymer. The aqueous admixture is formed into a desired physical shape, such as by casting a sheet of the solution. The sheet is then dried to form a self-supporting film. Cross-linking is then effected by immersing the film in aqueous acid solution. The resultant product has excellent properties for use as a battery separator, including a low electrical resistivity, the value of which is principally determined by the amount of cross-linking which is easily and closely controlled by the amount of cross-linking agent added to the polymer.

A technique for preparing Beauveria spp. for scanning electron microscopy.

Abstract: Vapor fixation for 96 h with 1% osmium tetroxide (OsO4) and 3–4 days air drying produced distortion-free specimens of Beauveria spp for examination with the scanning electron microscope A combination of 4 h OsO4 vapor fixation and freeze-drying also reduced disruption satisfactorily but specimens were not as well preserved as with the first method Preparation methods that were ineffective in preventing collapse of hydrophilic structures were Cling Free® sprayed on specimens prior to examination, freeze-drying, critical-point drying (of unfixed material), and vapor fixation with glutaraldehyde

Comparison of the activities and stabilities of alkaline glutaraldehyde sterilizing solutions.

Abstract: The sporicidal activities and shelf life stabilities of three alkaline glutaraldehyde-containing sterilizing solutions were evaluated. The time required to reduce a population of spores by one log is the D-value. The test methods employed were the D-value and survival rates. Sporicidin, a new preparation containing a phenolic buffer, was both more stable and more active against test spores than two other test agents, Cidex and Cidex-7.

Studies on the immobilization of trypsin on sand

Abstract: Trypsin was immobilized on sand using five different methods. Attempts were made to attach amino-functional groups onto sand using 3-aminopropyltriethoxysilane, hexamethylenetetramine, hexamethylenediamine, and melamine. Glutaraldehyde was used as a bifunctional agent in all the methods. Methods for the estimation of the proteolytic 1activity and protein content of immobilized trypsin were standardized. The maximum retained activity was observed for trypsin immobilized on sand via 3-aminopropytriethoxysilane and glutaraldehyde. Immobilized trypsin showed a shift in the pH optimum toward the acidic side over that of soluble trypsin in all five cases. The optimum temperature for both native and immobilized trypsin prepared by the silane-glutaraldehyde method was found to be 45°C. However, the pH and thermal stabilities of immobilized trypsin were observed to be better than that of the native enzyme.

Immobilization of beta-galactosidase and other enzymes onto p-amino-carbanilated cellulose derivatives.

Abstract: β-Galactosidase and other enzymes were immobilized on p-amino-carbanilated derivatives of cellulose and methylol cellulose using the diazo method and through glutaraldehyde. The optimum conditions for coupling cellulose tri-(p-amino-carbanilate) (CTAC) to β-galactosidase were established. The diazo coupling method with CTAC gave greater activity than with glutaraldehyde when coupled to β-galactosidase (Escherichia coli). The stability of the CTAC–β-galactosidase system was examined. The disubstituted p-amino-carbanilate derivative (CDAC) gave a lower activity, whereas the methylol analog (MCTAC) gave slightly greater activity. The CTAC was also used to immobilize glucose oxidase, trypsin, pepsin, and papain.

Effects of glutaraldehyde and glycerol on freeze-fractured Escherichia coli

Abstract: Glutaraldehyde and glycerol are widely used in the freeze-fracture technique as sample pretreatments before rapid freezing. However, they can both introduce relevant structural changes and influence the visualization of the fracture faces and surfaces of membranes. A comparison of the results obtained on E. coli cells differently pretreated with glutaraldehyde and glycerol is presented. In particular the effect on the distribution and density of the intramembranous particles (IMP) is pointed out. Glycerol treatment at 310 K introduces an IMP redistribution, outlined by the appearance of several smooth areas on the fracture faces of the cytoplasmic membrane, which is prevented by glutaraldehyde prefixation at the same temperature. On the other hand, glutaraldehyde treatment at 310 K following glycerol incubation results in the disappearance of the smooth areas, suggesting a substantial change in the IMP distribution caused by the fixative. Cells shifted down to 277 K and treated with glycerol at this temperature before quick-freezing, present on the convex fracture face of the cytoplasmic membrane large smooth areas resulting from a lipid transition while the smaller areas observed at 310 K are not detectable. Glutaraldehyde treatment at 277 K seems also to be responsible for a redistribution of IMP since poorly delimited large smooth areas, containing several IMP, can be observed. In this paper the results of a statistical analysis are also reported, showing that the IMP density can be strongly influenced by pretreatments.

Structure of the nucleoid in cells of Streptococcus faecalis.

Abstract: The structure of the nucleoid of Streptococcus faecalis (ATCC 9790) was examined and compared in the unfixed and fixed states by immersive refractometry and electron microscopy. It appears from these studies that the nucleoid structure is much more centralized in unfixed chloramphenicol-treated (stationary-phase) cells than it is in cells in the exponential phase of growth. The more dispersed configuration of the exponential-phase nucleoid could be preserved by fixation in glutaraldehyde, but not in Formalin or in osmium tetroxide. One important factor in explaining these differences in preservation is that glutaraldehyde (but not Formalin or osmium tetroxide) can rapidly cross-link the amino groups of macromolecules in cells. It was also observed that osmium tetroxide resulted in a preferential breakdown of nascent ribonucleic acid. These results are interpreted as indicating that glutaraldehyde is able to stabilize the exponential-phase nucleoid before it assumes the more central appearance seen in osmium tetroxide- and Formalin-fixed cells. These results are discussed in terms of the proposed organization of the exponential-phase nucleoid in unfixed cells.

Effect of protein cross-linking reagents on membrane currents of squid axon.

Abstract: The effects of the protein cross-linking reagents glutaraldehyde, tannic acid, and formaldehyde were examined in voltage-clamped and internally perfused squid axons. All three reagents reduced sodium and potassium currents and reduced or abolished sodium inactivation. Glutaraldehyde and tannic acid produced an alteration of potassium current kinetics.

α-1,4-Glucosidase–albumin polymers : in vitro properties and advantages for enzyme replacement therapy

Abstract: Soluble polymers of rat (or human) albumin and α-1,4-glucosidase are prepared using the cross-linking agent glutaraldehyde. The resulting polymer has an average molecular weight of 800 000 indicating an average composition of 12 albumin molecules for each enzyme molecule. Compared with an equivalent amount of free enzyme, the enzyme–albumin polymer has an increased resistance to heat denaturation (half-life of 15 h compared with 1 h for free enzyme at 37 °C) and to proteolysis by trypsin (half-life of 180 min compared with 10 min). The high degree of resistance to bioinactivation of the enzyme–albumin polymer is discussed in relation to requirements for enzyme replacement therapy in a range of metabolic diseases including type II glycogenosis (Pompe's disease) where α-1,4-glucosidase is the defective enzyme.

Treatment with lithium salts reduces ethanol dehydration shrinkage of glutaraldehyde fixed tissue

Abstract: Changes in the area of glutaraldehyde fixed 15 day p.c. mouse embryo limbs were recorded using a Quantimet 720 image analysing computer attached to a light microscope: during a period of treatment with an isotonic salt solution (mostly halides of the alkali or alkaline earth metals); a subsequent wash with distilled water; and dehydration through a 30, 50, 70, 80, 90, and 100% ethanol series. Pretreatment with NaCl, KCl, RbCl had no significant effect. Treatment with LiCl, LiNO3, LiF (0.03 M), CsF and CsCl caused an increase (relative to Na, K or Rb treated samples) in the specimen volume during dehydration, which persisted in 100% ethanol. Li treated samples showed the largest post-critical-point-drying (CPD) volumes, followed by Cs treated tissue. Pretreatment with Be, Mg, Ca, Sr and Ba chlorides caused shrinkage and the 100% ethanol and post-CPD volumes of these samples were all lower than those treated with the monovalent cation containing salts.

The use of graft copolymers as enzyme supports

Abstract: The grafting of acrylonitrile on to nylon powder was carried out in DMF solution after vacuum impregnation. Initiation was radiation induced. A sample of graft copolymer was reduced and coupled to enzyme using a water soluble carbodi-imide, or using glutaraldehyde.

Immobilization of yeast cells on polyphenyleneoxide

Abstract: The potential of a polymeric product of 2, 6-dimethylphenol as a support for immobilized intact yeast cells was investigated. The procedure used is based on modification of the polymeric adsorbent by adsorption of glutaraldehyde, and the immobilization of cells is probably accomplished by their adsorption and covalent linkage.

Glutaraldehyde nonfixation of isolated viral and yeast RNAs.

Abstract: The RNAs of brome mosaic (BMV), barley stripe mosaic (BSMV), and tobacco mosaic (TMV) viruses were inactivated by reaction with buffered glutaraldehyde. Glutaraldehyde did not fix 4% BMV-RNA, 20% t-RNA, 5% polyadenylic acid, or 5% adenosine monophosphate into water-insoluble precipitates, or gels, in distilled water or in low or high ionic strength buffers nor did it change their ultraviolet (UV) spectra. Two SDS- and phenol-purified commercial yeast RNA preparations from different sources gave UV spectra typical of pure RNA, but could not be freed of a contaminant that reacted with glutaraldehyde by forming a precipitate. The yeast RNAs did not become water-insoluble after glutaraldehyde reaction. BMV-RNA precipitated by Mg2+ could not be cross-linked into an insoluble form by glutaraldehyde. Nonfixation of RNA by glutaraldehyde must be considered in interpretation of attempts to localize RNA by electron microscopy.

Effects of preparation and fixation on three quantitative fluorescent cytochemical procedures.

Abstract: A series of experiments was undertaken in which cells dissociated from the abdominal lymph nodes of mice were lightly centrifuged into slides and fixed either wet or after drying in 70% ethanol, 1% glutaraldehyde, 1% formaldehyde, or neutral formalin. Three fluorescent cytochemical methods were evaluated: staining of DNA with mithramycin; fluorochroming of basic groups of proteins with brilliant sulfaflavine (BSF); and staining of sulfhydryl and disulfide groups with N-(7-dimethylamino-4-methylcoumarinyl)maleimide (DACM). In the case of mithramycin, the best results were obtained after fixation in 70% ethanol without drying. Staining of dried preparations fixed in 1% glutaraldehyde also yielded reasonably consistent results, although the fluorescence was lower, and the variability higher, than in the group fixed without drying in 70% ethanol. The use of fixatives containing formaldehyde resulted in fluorescence values of only about onethird those of the other two groups, and the variability of the data was higher. In material stained with BSF, satisfactory results were obtained in preparations fixed without drying in neutral formalin containing mersalyl acid. Other fixatives could be used, but the resulting coefficients of variation were higher than those of formalin-fixed material. Sulfhydryl to disulfide ratios approaching those expected from biochemical evidence were obtained in DACM-stained material only after fixation without drying in neutral formalin containing mersalyl acid. Inverted sulfhydryl-disulfide ratios were observed in material fixed without drying in 70% ethanol; and in dried metarial fixed in 1% formaldehyde, neutral formalin, or 1% glutaraldehyde.

Process for immobilizing cells

Abstract: The method for immobilising cells on a surface entails binding the cells with the aid of a coupling agent to the surface of a plastic material, e.g. polyvinyl chloride or polystyrene. The plastic material is preferably used in the form of a microtitre plate. Preferably used as coupling agent is an aldehyde with at least 2 aldehyde groups, especially glutaraldehyde. The method for immobilising cells is particularly suitable for the determination of binding of antibodies.