Pfizer selective enterococcus agar overlay method for the enumeration of fecal streptococci by membrane filtration.
(33/892)
The use of Pfizer selective enterococcus (PSE) agar with the membrane filter technique for the enumeration of fecal streptococci is limited due to the inability of the characteristic black precipitate, indicative of esculin hydrolysis, to diffuse from the medium through the membrane. A modification of the membrane filter technique that consisted of placing the membrane on PSE agar and overlaying it with tempered PSE agar was evaluated by comparing recovery, selectivity, and other parameters with M-enterococcus and KF-streptococcus agars, two selective media routinely used with the membrane filter technique for the enumeration of fecal streptococci in water and wastewater. No statistically significant differences could be demonstrated in the recovery capabilities of the three media. Inasmuch as the PSE overlay technique requires only 24 h of incubation as opposed to 48 h for the other two media, this modification may have some merit in water pollution monitoring programs. (+info)
Immobilization of antibodies on ultraflat polystyrene surfaces.
(34/892)
BACKGROUND: Functional antibody surfaces were prepared on ultraflat polystyrene surfaces by physical adsorption, and the uniform distribution of monoclonal antibodies against hepatitis B surface antigen (anti-HBs) on such surfaces and the presence of dense hepatitis B surface antigen (HBsAg) particles captured by immobilized antibodies were identified. METHODS: A model polystyrene film was spin-coated directly onto a silicon wafer surface. Atomic force microscopy was used to directly monitor the immobilization of anti-HBs antibodies and their specific molecular interaction with HBsAg. Enzyme immunoassay was also used to characterize functional antibody surfaces. RESULTS: A mean roughness of 2 A for areas of 25 microm(2) was produced. We found a uniform distribution of anti-HBs antibodies on ultraflat polystyrene surfaces and the presence of dense HBsAg particles bound to such anti-HBs surfaces after incubation with HBsAg. CONCLUSIONS: This study confirmed the potential of preparing dense, homogeneous, highly specific, and highly stable antibody surfaces by immobilizing antibodies on polystyrene surfaces with controlled roughness. It is expected that such biofunctional surfaces could be of interest for the development of new solid-phase immunoassay techniques and biosensor techniques. (+info)
Effect of salivary secretory IgA on the adhesion of Candida albicans to polystyrene.
(35/892)
Attachment of Candida albicans to plastic materials of dental prostheses or to salivary macromolecules adsorbed on their surface is believed to be a critical event in the development of denture stomatitis. In an earlier study, it was shown that adhesion of C. albicans to polystyrene, a model system to study the adhesion of C. albicans to plastic materials, can be partially inhibited with an mAb directed against cell wall polysaccharides of C. albicans. In the present study, the role of whole saliva in the adhesion of C. albicans to polystyrene has been investigated, and three mAbs directed against epitopes of cell wall mannoproteins have been used to mimic the inhibitory effect observed with salivary secretory IgA (sIgA) on the adhesion of C. albicans to polystyrene. In the absence of whole saliva, adherence of C. albicans 3153 increased with germination. However, the presence of whole saliva enhanced the adhesion to polystyrene of C. albicans 3153 yeast cells but decreased the adhesion of germinated cells. The enhancement of adhesion of yeast cells to polystyrene mediated by saliva was confirmed with an agerminative mutant of C. albicans 3153. The inhibition of the adhesion of C. albicans 3153 germ tubes to polystyrene was due to the salivary sIgA since sIgA-depleted saliva enhanced the adhesion of C. albicans 3153 to polystyrene. The inhibitory effect mediated by sIgA was not related to the inhibition of germination but to the blockage of adhesins expressed on the cell wall surface of the germ tubes. The three mAbs studied reduced the adhesion of C. albicans 3153 to polystyrene at levels equivalent to those for purified sIgA. The highest reduction in the adhesion was obtained with the IgA mAb N3B. The best results were obtained when the three mAbs were combined. The results suggest that whole saliva plays a different role in the adhesion of C. albicans to polystyrene depending on the morphological phase of C. albicans. These results may give new insights into the conflicting role of saliva in the adhesion of C. albicans to plastic materials of dental prostheses. (+info)
A bead-based method for multiplexed identification and quantitation of DNA sequences using flow cytometry.
(36/892)
A new multiplexed, bead-based method which utilizes nucleic acid hybridizations on the surface of microscopic polystyrene spheres to identify specific sequences in heterogeneous mixtures of DNA sequences is described. The method consists of three elements: beads (5.6-microm diameter) with oligomer capture probes attached to the surface, three fluorophores for multiplexed detection, and flow cytometry instrumentation. Two fluorophores are impregnated within each bead in varying amounts to create different bead types, each associated with a unique probe. The third fluorophore is a reporter. Following capture of fluorescent cDNA sequences from environmental samples, the beads are analyzed by flow cytometric techniques which yield a signal intensity for each capture probe proportional to the amount of target sequences in the analyte. In this study, a direct hybrid capture assay was developed and evaluated with regard to sequence discrimination and quantitation of abundances. The target sequences (628 to 728 bp in length) were obtained from the 16S/23S intergenic spacer region of microorganisms collected from polluted groundwater at the nuclear waste site in Hanford, Wash. A fluorescence standard consisting of beads with a known number of fluorescent DNA molecules on the surface was developed, and the resolution, sensitivity, and lower detection limit for measuring abundances were determined. The results were compared with those of a DNA microarray using the same sequences. The bead method exhibited far superior sequence discrimination and possesses features which facilitate accurate quantitation. (+info)
Characterization and optimization of bovine Echinococcus granulosus cyst fluid to be used in immunodiagnosis of hydatid disease by ELISA.
(37/892)
The aim of this work was to assess the influence in the diagnostic value for human hydatid disease of the composition of bovine hydatid cyst fluid (BHCF) obtained from fertile (FC) and non-fertile cysts (NFC). Eight batches from FC and 5 from NFC were prepared and analysed with respect to chemical composition: total protein, host-derived protein, carbohydrate and lipid contents. No differences were observed in the first two parameters but carbohydrate and lipid contents were shown to be higher in batches from FC than in those from NFC. Bands of 38 and 116 kD in SDS-PAGE profiles were observed to be present in BHCF from FC only. Two pools were prepared from BHCF batches obtained from FC (PFC) and NFC (PNFC), respectively. Antigen recognition patterns were analysed by immunoblot. Physicochemical conditions for adsorption of antigens to the polystyrene surface (ELISA plates) were optimized. The diagnostic value of both types of BHCF as well as the diagnostic relevance of oxidation of their carbohydrate moieties with periodate were assessed by ELISA using 42 serum samples from hydatid patients, 41 from patients with other disorders, and 15 from healthy donors. Reactivity of all sera against native antigen were tested with and without free phosphorylcholine. The best diagnostic efficiency was observed using BHCF from periodate-treated PFC using glycine buffer with strong ionic strength to coat ELISA plates. (+info)
Impact of the agr quorum-sensing system on adherence to polystyrene in Staphylococcus aureus.
(38/892)
Biofilm formation by Staphylococcus aureus is a serious problem in nosocomial infections. There are great differences in the capacity of S. aureus to express biofilms, but the reasons are unknown. In all, 105 S. aureus strains were tested for a correlation between the agr quorum-sensing system phenotype and the ability of S. aureus to adhere to polystyrene. Some 78% of agr-negative, but only 6% of agr-positive, strains formed a biofilm, demonstrating a profound impact of agr on biofilm formation. This result was confirmed with defined agr mutants and by inhibition of agr with quorum-sensing blockers. The observed effect was not due to differential expression of the autolysin Atl or of the exopolysaccharide polysaccharide intercellular adhesin but seemed to be caused, at least in part, by the surfactant properties of delta-toxin. The detected biofilm-enhancing effect of S. aureus quorum-sensing blockers call into question the proposed therapeutic use of such substances. (+info)
End-specific covalent photo-dependent immobilisation of synthetic DNA to paramagnetic beads.
(39/892)
A novel approach for light-dependent covalent immobilisation of synthetic DNA oligomers to amino-coated paramagnetic beads is described. A hetero-bifunctional photo-reactive cross-linking chemical, 4-nitrophenyl 3-diazopyruvate, is applied to attach 5' amino-modified DNA to both silica and polystyrene paramagnetic beads. The coupling yields are comparable with similar methods in which no photo-reactive chemicals are used. The immobilised DNA on the polystyrene and silica beads was used efficiently in hybridisation experiments. An extension of this approach to light-directed immobilisation of specific DNA to beads, located at different positions in micro-flow reactors, opens up a range of integrated applications to complex diagnostics, evolutionary biotechnology and novel areas such as DNA computing. (+info)
Cytochemical demonstration of hydrogen peroxide in polymorphonuclear leukocyte phagosomes.
(40/892)
Phagocytosis by polymorphonuclear leukocytes (PMN) is accompanied by specific morphological and metabolic events which may result in the killing of internalized micro-organism. Hydrogen peroxide is produced in increased amounts during phagocytosis (17) and in combination with myeloperoxidase and halide ions constitute a potent, microbicidal mechanism (8,9,11). There can be direct iodination of micro-organisms (10), or alternatively, other intermediate reaction products, i.e. chloramines and aldehydes (21), can exert a microbicidal effect. The H2O2-peroxidase-halide system is presumed to operate within the phagocytic vacuole (12,18). Myeloperoxidase, present in the primary granules of PMN, enters the phagocytic vacuole during degranulation (1,4,7), and halide ions are probably derived from the extracellular medium or are present in the PMN (see 11, 18). For the operation of this system in intact cells, the presence of H2O2 in the phagocytic vacuole is necessary, and indeed this has been suggested by the work of several investigators (12, 18, 21). In the present investigation, the diaminobenzidine reaction of Graham and Karnovsky (5), modified to utilize endogenous myeloperoxidase and hydrogen peroxide, has been applied to actively phagocytizing PMN to demonstrate cytochemically the presence of H2O2 in the phagocytic vacuole. (+info)