Sensitive counting of viable Enterobacteriaceae in seawaters and relationship with fecal indicators.

A fluorescent in situ hybridization based assay was used to enumerate viable Enterobacteriaceae members in seawaters by solid phase cytometry. The method was specific, highly sensitive (1cell/100ml) and allowed the quantification of VNC Enterobacteriaceae cells during an osmotic stress. Investigations on contaminated coastal seawater revealed a strong correlation between Enterobacteriaceae counts and standard fecal indicators.

Application of laser scanning cytometry followed by epifluorescent and differential interference contrast microscopy for the detection and enumeration of Cryptosporidium and Giardia in raw and potable waters.

Aims: The main goal of this study was to validate a new laser scanning cytometry method (ChemScanRDI) that couples immunofluorescence detection with differential interference contrast (DIC) confirmation, against manual microscopic enumeration of Giardia and Cryptosporidium (oo)cysts. This study also assessed the basic performance of the new Association Française de Normalisation (AFNOR) NF T 90-455 method for Giardia and Cryptosporidium (oo)cyst enumeration with respect to (oo)cyst yield, linearity, repeatability, influence of turbidity and detection limit in raw and potable waters.

Methods And Results: The new standard method relies on cartridge (Envirocheck) filtration, immunomagnetic separation purification, immunofluorescence staining and detection followed by DIC confirmation.

Rapid detection and enumeration of Naegleria fowleri in surface waters by solid-phase cytometry.

A new method for the rapid and accurate detection of pathogenic Naegleria fowleri amoebae in surface environmental water was developed. The method is based on an immunofluorescent assay combined with detection by solid-phase cytometry. In this study we developed and compared two protocols using different reporter systems conjugated to antibodies.

Enumeration of total viable microorganisms in an antibiotic raw material using ChemScan solid phase cytometer.

A preliminary study was performed for the enumeration of microorganism contamination of the macrolide antibiotic, Spiramycin, using epifluorescence with the ChemScan solid phase cytometer. The artificial spiking of Spiramycin powder antibiotic with pure culture of four microorganisms led to complete recovery of the tested organisms, whatever their sensitivity to the bacteriostatic activity of the drug. With the conventional plate method run in parallel, complete recovery was only obtained for Spiramycin resistant organisms.

Nonanoic Acid, a Fungal Self-Inhibitor, Prevents Germination of Rhizopus oligosporus Sporangiospores by Dissipation of the pH Gradient.

Germination of Rhizopus oligosporus sporangiospores is characterized by swelling of the spores and subsequent emergence of germ tubes. Changes in spore morphology and alterations in intracellular pH (pH(infin)) of the sporangiospores were assessed during the germination process by flow cytometry. Sporangiospores were stained with carboxyfluorescein by incubation with carboxyfluorescein diacetate.

A Novel Method for Continuous Determination of the Intracellular pH in Bacteria with the Internally Conjugated Fluorescent Probe 5 (and 6-)-Carboxyfluorescein Succinimidyl Ester.

A novel method based on the intracellular conjugation of the fluorescent probe 5 (and 6-)-carboxyfluorescein succinimidyl ester (cFSE) was developed to determine the intracellular pH of bacteria. cFSE can be taken up by bacteria in the form of its diacetate ester, 5 (and 6-)-carboxyfluorescein diacetate succinimidyl ester, which is subsequently hydrolyzed by esterases to cFSE in the cytoplasm. When Lactococcus lactis cells were permeabilized with ethanol, a significant proportion of cFSE was retained in the cells, which indicated that cFSE was bound intracellularly.

Characterization of uptake and hydrolysis of fluorescein diacetate and carboxyfluorescein diacetate by intracellular esterases in Saccharomyces cerevisiae, which result in accumulation of fluorescent product.

Flow cytometry is a rapid and sensitive method which may be used for the detection of microorganisms in foods and drinks. A key requirement for this method is a sufficient fluorescence staining of the target cells. The mechanism of staining of the yeast Saccharomyces cerevisiae by fluorescein diacetate (FDA) and 5- (and 6-)carboxyfluorescein diacetate (cFDA) was studied in detail.

Energy-dependent, carrier-mediated extrusion of carboxyfluorescein from Saccharomyces cerevisiae allows rapid assessment of cell viability by flow cytometry.

Carboxyfluorescein diacetate is a nonfluorescent compound which can be used in combination with flow cytometry for vital staining of yeasts and bacteria. The basis of this method is the assumption that, once inside the cell, carboxyfluorescein diacetate is hydrolyzed by nonspecific esterases to produce the fluorescent carboxyfluorescein (cF). cF is retained by cells with intact membranes (viable cells) and lost by cells with damaged membranes.

Application of flow cytometry to rapid microbial analysis in food and drinks industries.

In food and drinks industries, the time required for conventional tests can lead to substantial delays in product release to the market. Flow cytometry (FCM) has been used in conjunction with viability markers for rapid counting of yeast, mould and bacterial cells in food products. A single-parameter flow cytometer has proved applicable to the rapid detection of low numbers of microbial contaminants in finished products.

Rapid detection of members of the family Enterobacteriaceae by a monoclonal antibody.

Six monoclonal antibodies directed against enterobacteria were produced and characterized. The specificity of one of these antibodies (CX9/15; immunoglobulin G2a) was studied by indirect immunofluorescence against 259 enterobacterial strains and 125 other gram-negative bacteria. All of the enterobacteria were specifically recognized, the only exception being Erwinia chrysanthemi (one strain tested).

Enzymatic polymerization of 5-mercuriuridine-5'-diphosphate with polynucleotide phosphorylase from E. coli.

We report the polymerization of 5-mercuriuridine-5'-diphosphate (ppUHgX), in the presence of an excess of beta-mercaptoethanol, with polynucleotide phosphorylase from E. coli. A degradation of mercurated nucleotides with mercaptans was observed and about 30% of incorporation of mercury in the polymer was obtained after treatment with pancreatic RNase.

Deoxyribonucleic acid-protein and deoxyribonucleic acid interstrand cross-links induced in isolated chromatin by hydrogen peroxide and ferrous ethylenediaminetetraacetate chelates.

DNA-protein and DNA interstrand cross-links were induced in isolated chromatin after treatment with H2O2 and ferrous ethylenediaminetetraacetate (EDTA). Retention of DNA on membrane filters after heating of chromatin in a dissociating solvent indicated the presence of a stable linkage between DNA and protein. Treatment of protein-free DNA with H2O2/Fe2+-EDTA did not result in enhanced filter retention.

Structural requirements of (2'-5') oligoadenylate for protein synthesis inhibition in human fibroblasts.

The structural requirements of (2'-5')-oligoadenylic acid (pppA(2'p5'A)x, X greater than or equal to 1 or (2'-5'An) for inhibition of protein synthesis in cells were examined with a modified calcium-coprecipitation technique, using a series of trinucleotide analogs (pppA2'p5'A2'p5'N, N=rC, rG, rU, T, dC, dG, dA). In this system both the degree and the duration of the inhibition of protein synthesis were dependent on the added concentration of (2'-5')A3. Of all the heterotrimers, only the deoxy A derivative was active as an inhibitor of protein synthesis, while the other members of the analog series were found to have no inhibitory effects.

Blue dextran Sepharose chromatography of the tryptophanyl-tRNA synthetase of E. coli: a potential application for the purification of the enzyme.

E. coli tryptophanyl-tRNA synthetase can form a complex with Blue-dextran Sepharose, in the presence or in the absence of Mg++. In its absence, the complex is dissociated by either ATP or cognate tRNATrp.

Blue-dextran--Sepharose affinity chromatography: recognition of a polynucleotide binding site of a protein.

Native Escherichia coli polynucleotide phosphorylase can be retained on blue-dextran--Sepharose. The bound enzyme cannot be displaced by its mononucleotide substrates such as ADP, UDP, CDP, GDP and IDP, but it is easily eluted by its polymeric substrates. Under identical conditions, lactate dehydrogenase, bound on blue-dextran--Sepharose, is not eluted by poly(I) but can be specifically displaced by NADH.

Immunochemical measurement of conformational heterogeneity of poly(inosinic acid).

Several pure poly(I) preparations differed in: (a) their complement fixation reactivity with anti-poly(I) antiserum; (b) their ability to bind to a solid-phase anti-poly(I) antibody-Sepharose column; (c) their ability to inactivate serum complement; and (d) their reactivity with purified antibodies to double-stranded RNA. In particular, poly(I) samples that could induce interferon production differed from non-inducer poly(I)s; the inducers reacted weakly with anti-poly(I) antiserum and were the only ones that reacted with antibodies to double-stranded RNA. One inducer poly(I) did not inactivate complement, and differed from non-inducer poly(I) in quantitative aspects of poly(I) .

Poly-2'-deoxy-2'-fluoro-cytidylic acid: enzymatic synthesis, spectroscopic characterization and interaction with poly-inosinic acid.

The polymerization of 2'deoxy-2'-fluoro-cytidine-diphosphate (dCflDP) by polynucleotide phosphorylase is barely detectable in the presence of Mg++ under usual experimental conditions for polymerization of nucleoside diphosphates. High concentrations of enzyme have to be used to accomplish the synthesis. Mn++ is a better activator than Mg++ for the reaction.

Antibodies to adenosine 5'-monophosphate: purification and specificity.

Antibodies to adenosine-5'-monophosphate were produced in rabbits by injecting a conjugate of the nucleotide (oxidized with periodate) with bovine serum albumin. Nucleotide-specific antibodies were isolated by affinity chromatography on oligoadenylic acids/agarose column. Pure immunoglobulin G antibodies were obtained by gel filtration on Sephadex G-200.