A Quantitative Evaluation of the Antifungal Properties of Glutaraldehyde

Fungistatic data were obtained from measurements of mycelial growth of several fungal species in the presence of acid or alkaline glutaraldehyde. Alkaline glutaraldehyde in concentrations > 0·1% (w/v) prevented growth of all species examined while 0·5% (w/v) acid glutaraldehyde was necessary to achieve this effect. Further fungistatic data were obtained using a Coulter Counter method to measure spore swelling. Fungicidal determinations resulted in a 99·99% reduction in viable count after 90 min contact with 0·5% (w/v) alkaline glutaraldehyde. A considerable drop in spore production was also observed after treatment with alkaline glutaraldehyde.     

Poly(3-hydroxybutyrate)/chitosan/ketoprofen or piroxicam composite microparticles: Preparation and controlled drug release evaluation

Poly(3-hydroxybutyrate)/chitosan/piroxicam or ketoprofen composite microparticles were prepared by the solid-in-water-in-oil emulsion-solvent evaporation technique with the aim of reducing the burst effect and controlling the drug release. Reservoir-type microparticles, composed of poly(3-hydroxybutyrate) microspheres embedded in a chitosan matrix were prepared. The size and morphological characteristics of the composite microparticles were evaluated in relation to the chitosan concentration and cross-linking with glutaraldehyde. Reservoir-type composite microparticles were obtained using 2.0% and 3.0% w/v chitosan solutions. A significant reduction in the burst effect and prolonged drug release were observed, particularly when higher chitosan and glutaraldehyde concentrations were used.     

L-DOPA production from tyrosinase immobilized on nylon 6,6

The production of L-DOPA immobilized on chemically modified nylon 6,6 membranes was studied in a batch reactor. Tyrosinase was immobilized on nylon using glutaraldehyde as a crosslinking agent. The effects of membrane pore size and glutaraldehyde concentration upon enzyme uptake and L-DOPA production were investigated. Enzyme uptake was unaffected by glutaraldehyde concentration; approximately 70% uptake was observed when 25% w/v (group 1), 5% (group 2), and 3% (group 3) glutaraldehyde were used, indicating that glutaraldehyde was in excess. Similarly, uptake was the same for membranes with 0.20 and 10 mum pore sizes.Membranes produced using different levels of glutaraldehyde exhibited dramatically different capacities for L-DOPA production, despite the fact that enzyme uptake was equivalent. Membranes from groups 2 and 3 (5% and 3% glutaraldehyde) produced L-DOPA at a rate of 1.70 mg L(-1) h(-1) over 170 h in a 500-mL batch reactor. However, no free L-DOPA was detected when group 1 membranes were used. Experimental evidence suggests that L-DOPA was produced, but remained bound to these membranes via excess glutaraldehyde left over from the immobilization process. Membrane pore size also effected L-DOPA production; less production was observed when 10-mum membranes were used, despite equivalent enzyme uptake. The observed difference in production may be due to differences in the pore density on the two types of membranes which could affect the access of the substrate to the immobilized enzyme.The results of these studies indicate that tyrosinase can be effectively immobilized on nylon 6,6. L-DOPA production was optimal when 0.20-mum-pore-size membranes were activated with 3-5% glutaraldehyde. Stability studies indicated a 20% reduction in activity over 14 days when the immobilized enzyme was used under turnover conditions. (c) 1996 John Wiley & Sons, Inc.     

Kinetics of glutaraldehyde fixation of erythrocytes: size, deformability, form, osmotic and hemolytic properties.

Red blood cells interact with glutaraldehyde (GA) in a complex kinetic pattern of events. At a given GA concentration in phosphate buffered saline (PBS), the sequence of cell 'volume' response, as measured by resistive pulse spectroscopy (RPS), includes: an immediate response to the overall solution osmolality; a constant volume, latent phase; a rapid swelling phase; an intermediate constant volume phase; and a shrinkage phase to a final steady state volume. The final volume depends on fixative solution osmolality; for GA concentrations between 0.05% and 0.25% w/v, fixative osmolalities of less than 355 mosM, including 'isotonic', or greater than 355 mosM, lead to final cell volumes greater or less than native, respectively. Cell-membrane deformability decreases continuously and monotonically with time, as assessed by RPS. The rate of fixation is a direct function of GA concentration, in accordance with a derived empirical expression. The measured kinetic responses are related to considerations of cell size, deformability, and form, and to mechanisms involved in abrupt osmotic hemolysis.     

α-Amylase immobilization on gelatin: Optimization of process variables

α-Amylase was extracted and purified from soybean seeds to apparent homogeneity by affinity precipitation. The homogeneous enzyme preparation was immobilized on gelatin matrix using glutaraldehyde as an organic hardener. Response surface methodology (RSM) and 3-level-3-factor Box–Behnken design was employed to evaluate the effects of immobilization parameters, such as gelatin concentration, glutaraldehyde concentration and hardening time on the activity of immobilized α-amylase. The results showed that 20% gelatin (w/v), 10% glutaraldehyde (v/v) and 1 h hardening time yielded an optimum immobilization of 82.5%.     

Oxidation of methane by Methylomicrobium album and Methylocystis sp. in the presence of H2S and NH3

Oxidation of methane by methanotrophs, Methylomicrobium album and Methylocystis sp., was measured at several initial concentrations of H₂S and NH₃ in the headspace of stoppered flasks, at the same initial concentration of methane as sole carbon and energy source: 15 % (v/v). No effect was observed at 0.01 % (v/v) H₂S and 0.025 % (v/v) NH₃ in gas phase but over 0.05 and 0.025 % (v/v), respectively, they inhibited the oxidation of methane. The effect of H₂S was stronger in Methylocystis sp. and both microorganisms were similarly affected by NH₃. Depending on their concentrations in gas phase, H₂S and NH₃ can thus affect the rate of oxidation of methane and biomass growth of both methanotrophs.     

Nutritional evaluation of Cyanobacterium (Nostoc sp.) extract in Rhizobium cultures

Extracts from Nostoc sp. biomass (CE) were compared with yeast extract (YE) in the formulation of media for cultivation of Rhizobium etli and Bradyrhizobium japonicum. Growth depended on the rhizobium strain and also on the type and concentration of the extract. The growth kinetics of R. etli were substantially influenced by the addition of CE an YE at concentrations between 0.3 and 1% (w/v); cultures containing YE showed shorter lag periods and higher growth rates and those with CE presented higher growth yields. Depending on the extracts, a different behaviour was displayed by the cultures at the stationary phase; cell viability increased or remained fairly constant in cultures developed on CE, and those developed on high concentrations of YE showed a decline in viable cell counts.     

Author index volume 65

Spores of Bacillus subtilis 168 were apparently fully inactivated by exposure to 2% (w/v) glutaraldehyde for 20 h but a few spores could be revived by further treatment with 10-100 mM NaOH. A similar effect was found with spores from a range of Bacillus species. A minimum concentration of 5% (w/v) glutaraldehyde was required to prevent the alkali-induced reactivation. The implications of these results for the use of glutaraldehyde as a sporicidal agent are discussed.     

Nutritional evaluation of Cyanobacterium (Nostoc sp.) extract in Rhizobium cultures

Extracts from Nostoc sp. biomass (CE) were compared with yeast extract (YE) in the formulation of media for cultivation of Rhizobium etli and Bradyrhizobium japonicum. Growth depended on the rhizobium strain and also on the type and concentration of the extract. The growth kinetics of R. etli were substantially influenced by the addition of CE an YE at concentrations between 0.3 and 1% (w/v); cultures containing YE showed shorter lag periods and higher growth rates and those with CE presented higher growth yields. Depending on the extracts, a different behaviour was displayed by the cultures at the stationary phase; cell viability increased or remained fairly constant in cultures developed on CE, and those developed on high concentrations of YE showed a decline in viable cell counts.     

Biofixation of carbon dioxide by Spirulina sp. and Scenedesmus obliquus cultivated in a three-stage serial tubular photobioreactor

The increase in the concentration of atmospheric carbon dioxide is considered to be one of the main causes of global warming. As estimated by the Intergovernmental Panel on Climate Change (IPCC) criteria, about 10-15% of the gases emitted from the combustion coal being in the form of carbon dioxide. Microalgae and cyanobacteria can contribute to the reduction of atmospheric carbon dioxide by using this gas as carbon source. We cultivated the Scenedesmus obliquus and Spirulina sp. at 30 degrees C in a temperature-controlled three-stage serial tubular photobioreactor and determined the resistance of these organisms to limitation and excess of carbon dioxide and the capacity of the system to fix this greenhouse gas. After 5 days of cultivation under conditions of carbon limitation both organisms showed cell death. Spirulina sp. presenting better results for all parameters than S. obliquus. For Spirulina sp. the maximum specific growth rate and maximum productivity was 0.44 d(-1), 0.22 g L(-1)d(-1), both with 6% (v/v) carbon dioxide and maximum cellular concentration was 3.50 g L(-1) with 12% (v/v) carbon dioxide. Maximum daily carbon dioxide biofixation was 53.29% for 6% (v/v) carbon dioxide and 45.61% for 12% carbon dioxide to Spirulina sp. corresponding values for S. obliquus being 28.08% for 6% (v/v) carbon dioxide and 13.56% for 12% (v/v) carbon dioxide. The highest mean carbon dioxide fixation rates value was 37.9% to Spirulina sp. in the 6% carbon dioxide runs.     

Growth behaviour and bioproduction of indole acetic acid by a Rhizobium sp. isolated from root nodules of a leguminous tree Dalbergia lanceolaria

The Rhizobium sp. isolated from healthy and mature root nodules of a leguminous tree, Dalbergia lanceolaria Linn. f., preferred mannitol and KNO3 for growth as carbon and nitrogen sources, respectively. The bacterium produced a high amount (22.3 microg/ml) of indole acetic acid (IAA) from L-tryptophan supplemented basal medium. Growth and IAA production started simultaneously. IAA production was maximum at 20 hr when the bacteria reached the stationary phase of growth. Cultural requirements were optimized for maximum growth and IAA production. The IAA production by the Rhizobium sp. was increased by 270.8% over control when the medium was supplemented with mannitol (1%,w/v), SDS (1 microg/ml), L-asparagine (0.02%,w/v) and biotin (1 microg/ml) in addition to L-tryptophan (2.5 mg/ml). The possible role of IAA production in the symbiosis is discussed.     

Growth of food-borne pathogenic bacteria in oil-in-water emulsions: II—Effect of emulsion structure on growth parameters and form of growth

The growth rates and yields of Listeria monocytogenes and Yersinia enterocolitica were determined in liquid culture media, and in model oil-in-water emulsions that contained 30, 70 or 83% (v/v) hexadecane. In emulsions with a mean droplet size of 2 μm containing 83% (v/v) hexadecane, the growth of both organisms resulted in decreased yields. Additionally, in these emulsions adjusted to pH 5·0 or 4·4 the growth rate of L. monocytogenes was significantly less than in other model systems which had an aqueous phase of equivalent chemical composition. Microscopic examination of the 83% (v/v) emulsion showed that its microstructure immobilized the bacteria, which were constrained to grow as colonies. Bacteria behaved similarly in model emulsions of either hexadecane or sunflower oil. Manipulation of the droplet size distribution of the emulsions changed the form and rate of growth of bacteria within them.     

Uptake of l-[14C]-alanine by glutaraldehyde-treated and untreated spores of Bacillus subtilis

The effect of glutaraldehyde on the uptake of L-alanine, and subsequent germination, in spores of Bacillus subtilis NCTC 8236 was examined. Germination was induced by single amino acids, D-glucose and phosphate buffer at 37 degrees C. L-alanine was the best germinant of all amino acids tested. Pretreatment of spores with low concentrations of acid and alkaline glutaraldehyde inhibited subsequent germination, complete inhibition being observed at concentrations of 0.1% (w/v). This concentration also prevented the loss of heat resistance of spores placed in germination medium and exposed to 75 degrees C. Radioactive studies indicated that maximum uptake of L-alanine occurred after ca 30 min at 37 degrees C. Only 1.2% of available L-alanine was taken up during germination. Pretreatment of spores with glutaraldehyde did not interfere with L-alanine uptake at aldehyde concentrations up to 0.5% (w/v). However, this was significantly reduced at a glutaraldehyde concentration of 1.0% (w/v). Minimal differences were observed between acid and alkaline forms of the aldehyde. The results are discussed in terms of the mode of action of glutaraldehyde.     

Effect of polyols on the post-thawing motility of pellet-frozen ram spermatozoa

The cryoprotective effects of polyols in the absence and presence of glycerol in Tris-glucose-egg yolk based diluents on the post-thawing motility and acrosome integrity of pellet-frozen ram spermatozoa were examined. Incorporation of adonitol or xylitol (low molecular weight polyols; LMWPs) in diluents improved motility of spermatozoa in the absence of glycerol with maximum motility at 0.3 M (26.9 vs 13.3% mean motile spermatozoa; P < 0.001). Five concentrations of adonitol (0, 150, 300, 450, 600 mM) were examined in diluents containing 5 concentrations of glycerol (0, 1.5, 3.0, 4.5, 6.0% v/v). There was an additive effect of incorporation of 1.5% v/v glycerol with up to 450 mM adonitol (P < 0.001), but at higher levels of glycerol the incorporation of adonitol was detrimental to motility. The acrosome integrity of spermatozoa in diluents containing 0, 150 and 300 mM adonitol was superior to those containing 450 and 600 mM adonitol (46.1 vs 35.1% mean intact acrosomes; P < 0.05). Among the high molecular weight polyols (HMWPs) examined, better recovery of spermatozoa was obtained in diluents containing sorbitol than mannitol or inositol (P < 0.001). Sorbitol or mannitol (300 mM) improved the motility of spermatozoa in diluents without glycerol (P < 0.001), but the incorporation of HMWPs was detrimental in diluents containing glycerol. All five polyols were examined in isotonic diluents containing 360:0, 300:55, 240:110, 180:165, 120:220mM (Tris:polyol; 360 mosmol) and 6.0% v/v glycerol. There was a linear decrease in motility and acrosome integrity of spermatozoa with increasing polyol concentration in the diluent (P < 0.001) except for inositol, which was not detrimental. We conclude that the polyols examined have a cryoprotective effect on pellet-frozen ram spermatozoa except for inositol. However, in our study, no combination of polyols and glycerol was superior in terms of post-thawing motility and acrosome integrity of spermatozoa to 6.0% v/v glycerol alone in Tris-glucose-egg yolk diluents.     

Effect of sperm motility on human embryo quality in in vitro fertilization

Sperm motility is important for penetration of the zona pellucida, and this parameter has been reported to be the single most important factor determining fertilization rates. As there was no report on the relationship between sperm motility and embryo quality, we investigated the influence of sperm motility on embryo quality in 41 patients with tubal disease and/or obstruction. The patients were either unstimulated or stimulated with clomiphene or clomiphene and human menopausal gonadotrophin. Of 116 oocytes collected, 86 (74.1%) fertilised and cleaved; of these only 44 embryos had clear equal blastomeres without fragmentation (grade 3). Grade 3 embryos were equally distributed through all initial sperm motility categories, and through all categories of sperm concentration after swim-up. The ratio of motile sperm concentration in the initial semen sample to the final sperm concentration after swim-up varied from 0.5 to 67, and grade 3 embryos were distributed randomly from low to high ratios. The pregnancy rate in this series was only 14.6% per replacement. The rate of gestational sacs per embryo replaced was 7.0% (6/86); if “poor” embryos were excluded, the rate was 9.1% (6/66). The absence of correlation between sperm motility and embryo quality is discussed on morphological grounds.     

Sporicidal action of alkaline glutaraldehyde: factors influencing activity and a comparison with other aldehydes

The sporicidal efficacy of glutaraldehyde (2% w/v) was investigated under various conditions. Numerous factors influenced its activity: method of spore production, inherent spore resistance characteristics, alkalination, storage time and storage temperature. The sporicidal action of 2% alkaline glutaraldehyde at room temperature was compared with that of other aldehydes and commercially available formulations. Cidex (glutaraldehyde) and Sporicidin (glutaraldehyde + phenol full strength) were the most effective, followed by 8% (w/v) formaldehyde and 10% (v/v) Gigasept, a formaldehyde-containing product. Five per cent (v/v) Gigasept and 10% (w/v) glyoxal also had good sporicidal activity, though that of Sporicidin (1 : 16) was poor. No activity was observed with 10% (w/v) butyraldehyde.     

Extraction and electrospinning of gelatin from fish skin

Ultra-fine gelatin fibers were successfully fabricated by electrospinning from the solutions of Nile tilapia (Oreochromis niloticus) skin-extracted gelatin in either acetic acid or formic acid aqueous solutions. The extracted gelatin contained 7.3% moisture, 89.4% protein, 0.3% lipid, and 0.4% ash contents (on the basis of wet weight), while the bloom gel strength, the shear viscosity, and the pH values were 328 g, 17.8 mPa s, and 5.0, respectively. Both the acid concentration and the concentration of the gelatin solutions strongly influenced the properties of the as-prepared solutions and the obtained gelatin fibers. At low acid concentrations (i.e., 15% (w/v) extracted gelatin solutions in 10 and 20% (v/v) acetic acid solvents or 10-60% (v/v) formic acid solvents), a combination between smooth and beaded fibers was observed. At low concentrations of the gelatin solutions in either 40% (v/v) acetic acid solvent or 80% (v/v) formic acid solvent (i.e., 5-11%, w/v), either discrete beads or beaded fibers were obtained, while, at higher concentrations (i.e., 14-29%, w/v), only smooth or a combination of smooth and beaded fibers were obtained. The average diameters of the obtained fibers, regardless of the types of the acid solvents used, ranged between 109 and 761 nm. Lastly, cross-linking of the obtained gelatin fiber mats with glutaraldehyde vapor caused slight shrinkage from their original dimension, and the cross-linked gelatin fiber mats became stiffer.     

Sporicidal action of alkaline glutaraldehyde: factors influencing activity and a comparison with other aldehydes

The sporicidal efficacy of glutaraldehyde (2% w/v) was investigated under various conditions. Numerous factors influenced its activity: method of spore production, inherent spore resistance characteristics, alkalination, storage time and storage temperature. The sporicidal action of 2% alkaline glutaraldehyde at room temperature was compared with that of other aldehydes and commercially available formulations. Cidex (glutaraldehyde) and Sporicidin (glutaraldehyde + phenol full strength) were the most effective, followed by 8% (w/v) formaldehyde and 10% (v/v) Gigasept, a formaldehyde-containing product. Five per cent (v/v) Gigasept and 10% (w/v) glyoxal also had good sporicidal activity, though that of Sporicidin (1:16) was poor. No activity was observed with 10% (w/v) butyraldehyde.     

Immunogold localization of TGFβ 1 protein and mRNA in human skin using a colloidal gold/digoxygenin system

Tissue preservation, and immunogold cytochemical and in-situ hybridization labelling intensities vary according to the preparatory protocols used. We wished to determine which preparative protocols produce optimal preservation, protein and mRNA labelling. Nine combinations of fixative and embedding resin were therefore studied using postembedding immunoelectron microscopy and a novel immunogold digoxygenin in situ hybridization (ISH) system, to quantitate the presence of transforming growth factor beta 1 (TGF 1) protein and message in human skin. The best preservation was observed in tissue fixed in 1% glutaraldehyde and embedded in LR White resin or low acid glycolmethacrylate resin (LA-GMA). Preservation was poor in tissue fixed with 1% glutaraldehyde and fair in 4% paraformaldeyde, when embedded in Unicryl. Ethanediol dehydration coupled with LA-GMA embedding resulted in reasonable preservation. Based on quantitative measures of the labelling density for TGF 1 protein and mRNA, immunogold labelling was adequate with 1% glutaraldehyde fixation coupled with LR White or LA-GMA resins, and also with 4% paraformaldehyde and LR White resin, but was best with ethanediol dehydration and LA-GMA embedding. ISH labelling under basal conditions was best in LA-GMA with 1% glutaraldehyde or 4% paraformaldehyde. The ISH label in tissue fixed with 1% glutaraldehyde and embedded in LA-GMA was significantly increased by treatment with proteinase K. Overall, ethanediol dehydration was associated with a good immunoelectron microscopic (IEM) label while LA-GMA with 1% glutaraldehyde or 4% paraformaldehyde resulted in a consistently detectable ISH label. LA-GMA embedding with 1% glutaraldehyde fixation gave a good result with both IEM and ISH labelling.     

Effect of essential oils on pathogenic and biofilm-forming Vibrio parahaemolyticus strains

Abstract

In this study, the effect of three essential oils (EOs) – clove oil (CO), thyme oil (TO), and garlic oil (GO), which are generally recognized as safe – on the planktonic growth, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), motility, biofilm formation, and quorum sensing (QS) of Vibrio parahaemolyticus was investigated. All three EOs showed bacteriostatic activity, with MICs in the range 0.02%–0.09% (v/v). CO and TO completely controlled planktonic growth at 0.28% and 0.08% (v/v), which is four times their MIC (4?×?MIC), after 10?min, whereas GO completely controlled growth at 0.36% (v/v) (4?×?MIC) after treatment for 20?min. V. parahaemolyticus motility was significantly reduced by all three EOs at 4?×?MIC (0.28% for CO, 0.08% for TO, and 0.36% for GO), whereas QS was controlled and biofilm formation reduced by all three EOs at 8?×?MIC (0.56% for CO, 0.16% for TO, and 0.72% for GO) after 30?min of treatment. These results suggest that CO, TO, and GO have a significant inhibitory effect on V. parahaemolyticus cells in biofilm sand thus represent a promising strategy for improving food safety. These results provide the evidence required to encourage further research into the practical use of the proposed EOs in food preparation processes.