New insights for rapid evaluation of bactericidal activity: a semi-automated bioluminescent ATP assay.

Aims:   A new assay, much more rapid and efficient than the existing standardized tests, is introduced for the evaluation of bactericidal activity of chemical disinfectants and antiseptics under simulated practical conditions of use.

Methods And Results:   The bactericidal activity of biocides was quantified using a novel semi-automated assay based on the European Norm (EN) standard suspension tests but determining bacterial cell viability by intracellular adenosine tri-phosphate (ATP) content quantification instead of traditional culture-based microbiological techniques. The new test was validated by comparison to the standard suspension tests EN 1276 and EN 13727.

Differential persistence of F-specific RNA phage subgroups hinders their use as single tracers for faecal source tracking in surface water.

The four subgroups of F-specific RNA bacteriophages (I-IV) have been proposed as potential tracers for faecal source tracking. Groups II and III predominate in human sources while groups I and IV are most abundant in animal sources. The four subgroups of naturally occurring F-specific RNA bacteriophages were identified in different samples by plaque hybridization with genotype-specific probes and the persistence of each subgroup was evaluated.

Occurrence and distribution of culturable enteroviruses in wastewater and surface waters of north-eastern Spain.

Aims: Update information regarding occurrence and levels of culturable enteroviruses in several types of surface polluted waters in north-eastern Spain and determine the proportion of the different species and serotypes.

Methods And Results: The best procedures on hand in our laboratory for concentrating and quantifying culturable enteroviruses from different water sample types were used. Sequencing was used for typing the virus isolates.

A method to maintain mammalian cells for days alive at 4 degrees C.

This paper describes a method for the temporary storage of cultured cells. Cells from recently completed cell monolayers were trypsinized and then centrifuged. After centrifugation, the supernatant and pellet were kept at 4 degrees C for one week.

Evaluation of Escherichia coli host strain CB390 for simultaneous detection of somatic and F-specific coliphages.

Escherichia coli WG5, the strain recommended by the International Organization for Standardization (ISO) to detect somatic coliphages, was transformed to F(+) by introducing the plasmid Famp, which rendered it capable of simultaneously detecting both somatic and F-specific coliphages. Indeed, this strain, CB390, proved as effective in detecting similar numbers of phages as the sum of somatic and F-specific bacteriophages detected by the host strains recommended by both the ISO and the U.S.

A membrane-based quantitative carrier test to assess the virucidal activity of disinfectants and persistence of viruses on porous fomites.

A membrane-based quantitative carrier test method to assess the virucidal activity of disinfectants and the persistence of viruses on fomites under different environmental conditions is described. The method is based on the inactivation of the virus adsorbed to cellulose ester membranes followed by the direct enumeration of the viruses surviving the treatment without the need of an elution step. The method was suitable for four different human enteroviruses tested.

Enteroviruses and bacteriophages in bathing waters.

A new procedure for detecting and counting enteroviruses based on the VIRADEN method applied to 10 liters of seawater was examined. It improved the efficiency of detection by taking into account both the number of positive isolations and numbers found with traditional methods. It was then used to quantify viruses in bathing waters.

Double-layer plaque assay for quantification of enteroviruses.

We describe here a double-layer plaque assay for the quantification of enteroviruses, combining a monolayer plaque assay and a suspended-cell plaque assay. The double-layer assay provides significantly greater counts than other methods of virus quantification of both suspensions of pure culture viruses and naturally occurring viruses. The counts obtained by this method are approximately one order of magnitude greater than those obtained with the more commonly used method, the monolayer plaque assay.

Usefulness of different groups of bacteriophages as model micro-organisms for evaluating chlorination.

Aims: To assess the usefulness of bacterial and viral indicators in chlorination processes and to collect quantitative information necessary for risk assessment analysis in water disinfection processes based on chlorination.

Methods And Results: Naturally occurring bacterial indicators, bacteriophages and enteroviruses were determined to evaluate the effect of chlorination in groundwater and secondary sewage effluents. Additionally, the effect of chlorinating on selected bacteriophages, enteroviruses and Escherichia coli was also tested in spiked samples of bottled water and sewage effluents.

Bacterial host strains that support replication of somatic coliphages.

Somatic coliphages detected by Escherichia coli strain WG5 have been proposed as potential indicators of water quality. Their potential replication in the water environment is considered a drawback for their use as indicators. However, the contribution of replication outside the gut to the total numbers has never been quantified.

Comparison of polyvinylidene fluoride and polyether sulfone membranes in filtering viral suspensions.

Low-protein-binding membranes with a pore size of 0.22 microm are used to filter aqueous solutions containing viruses. Virus adsorption to the membranes is avoided if they are made of polyvinylidene fluoride (PVDF) or if they are made of cellulose esters saturated with beef extract.

Survival of bacterial indicator species and bacteriophages after thermal treatment of sludge and sewage.

The inactivation of naturally occurring bacterial indicators and bacteriophages by thermal treatment of a dewatered sludge and raw sewage was studied. The sludge was heated at 80 degrees C, and the sewage was heated at 60 degrees C. In both cases phages were significantly more resistant to thermal inactivation than bacterial indicators, with the exception of spores of sulfite-reducing clostridia.

Counting cytopathogenic virus adsorbed to cellulose nitrate membrane filters as a simple method for counting viruses in raw sewage and sewage effluents.

Using the VIRMETADEN (acronym derived from virus adsorption enumeration) method to count cytopathogenic viruses adsorbed to cellulose nitrate membrane filters from prefiltered and decontaminated sewage samples was shown to be feasible. The numbers of naturally occurring enteroviruses recovered by the VIRMETADEN method were significantly higher than those obtained by standard plaque assay. For prefiltration of sewage samples, low protein binding polyvinylidene fluoride (PVDF) membranes of 0.

New method for evaluation of virucidal activity of antiseptics and disinfectants.

Counting culturable viruses adsorbed to cellulose nitrate filters (the VIRADEN method) is proposed as a simple procedure for the evaluation of the virucidal activity of antiseptics and disinfectants. The virucidal activities of two different doses of iodine, chlorine, glutaraldehyde, and chlorhexidine digluconate on poliovirus 1 were tested with a standardized procedure and with the VIRADEN method. The two procedures assayed provided similar results.

A simple methodological approach for counting and identifying culturable viruses adsorbed to cellulose nitrate membrane filters.

We identified conditions under which Buffalo green monkey cells grew on the surfaces of cellulose nitrate membrane filters in such a way that they covered the entire surface of each filter and penetrated through the pores. When such conditions were used, poliovirus that had previously been adsorbed on the membranes infected the cells and replicated. A plaque assay method and a quantal method (most probable number of cytopathic units) were used to detect and count the viruses adsorbed on the membrane filters.