In microbiology the terms 'viability' and 'culturability' are often equated. However, in recent years the apparently self-contradictory expression 'viable-but-nonculturable' ('VBNC') has been applied to cells with various and often poorly defined physiological attributes but which, nonetheless, could not be cultured by methods normally appropriate to the organism concerned. These attributes include apparent cell integrity, the possession of some form of measurable cellular activity and the apparent capacity to regain culturability. We review the evidence relating to putative VBNC cells and stress our view that most of the reports claiming a return to culturability have failed to exclude the regrowth of a limited number of cells which had never lost culturability. We argue that failure to differentiate clearly between use of the terms 'viability' and 'culturability' in an operational versus a conceptual sense is fuelling the current debate, and conclude with a number of proposals that are designed to help clarify the major issues involved. In particular, we suggest an alternative operational terminology that replaces 'VBNC' with expressions that are internally consistent.

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Viability and activity in readily culturable bacteria: a review and discussion of the practical issues

We developed a protocol which yields purified bacterial DNA from the soil bacterial community. The bacteria were first dispersed and separated from soil particles in the presence of polyvinylpolypyrrolidone, which removes humic acid contaminants by adsorption to this insoluble polymer. The soil bacteria were then collected by centrifugation and lysed by using a comprehensive protocol designed to maximize disruption of the various types of bacteria present. Total bacterial DNA was purified from the cell lysate and remaining soil contaminants by using equilibrium density gradients. The isolated DNA was essentially pure as determined by UV spectral analysis, was at least 48 kilobases long, and was not subject to degradation, which indicated that there was no contaminating nuclease activity. The isolated DNA was readily digested by exogenously added restriction endonucleases and successfully analyzed by slot blot and Southern blot hybridizations. Using single-stranded, P-labeled DNA probes, we could detect and quantitate the presence of a specific microbial population in the natural soil community on the basis of the presence of a DNA sequence unique to that organism. The sensitivity of our methodology was sufficient to detect Bradyrhizobium japonicum at densities as low as 4.3 x 10 cells per g (dry weight) of soil, which corresponds to about 0.2 pg of hybridizable DNA in a 1-mug DNA sample.

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DNA Probe Method for the Detection of Specific Microorganisms in the Soil Bacterial Community

Enterococci are common, commensal members of gut communities in mammals and birds, yet they are also opportunistic pathogens that cause millions of human and animal infections annually. Because they are shed in human and animal feces, are readily culturable, and predict human health risks from exposure to polluted recreational waters, they are used as surrogates for waterborne pathogens and as fecal indicator bacteria (FIB) in research and in water quality testing throughout the world. Evidence from several decades of research demonstrates, however, that enterococci may be present in high densities in the absence of obvious fecal sources and that environmental reservoirs of these FIB are important sources and sinks, with the potential to impact water quality. This review focuses on the distribution and microbial ecology of enterococci in environmental (secondary) habitats, including the effect of environmental stressors; an outline of their known and apparent sources, sinks, and fluxes; and an overview of the use of enterococci as FIB. Finally, the significance of emerging methodologies, such as microbial source tracking (MST) and empirical predictive models, as tools in water quality monitoring is addressed. The mounting evidence for widespread extraenteric sources and reservoirs of enterococci demonstrates the versatility of the genus Enterococcus and argues for the necessity of a better understanding of their ecology in natural environments, as well as their roles as opportunistic pathogens and indicators of human pathogens.

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Enterococci in the Environment

Sampling and Data Treatment Methods Sampling Methods in Surface Waters, Munro Mortimer, Jochen F. Muller, and Matthias Liess Methods of Treatment of Data, Riccardo Leardi Radioanalytical Analysis Radioanalytical Methodology for Water Analysis, Jorge S. Alvarado Organoleptical Analysis Organoleptical Methodology, Abdul Moheman, Md. Musawwer Khan, and K. S. Siddiqi Analysis of Biological Parameters Bacteriological Analysis of Water, Paulinus Chigbu and Salina Parveen Marine Toxins Analysis, Luis M. Botana, A. Alfonso, M. R. Vieytes, N. Vilarino, A. M. Botana, C. Louzao, and C. Vale Algal Analysis, Leonardo Rubi Rorig Halogens, N-Compounds, and Phosphates Halogens, Geza Nagy and Livia Nagy Analysis of Sulfur Compounds in Water, Leo M. L. Nollet Determination of Ammonia in Water Samples, Juliana Antunes Galvao, Alexandre Matthiensen, Marilia Oetterer, Y. Moliner-Martinez, R. A. Gonzalez-Fuenzalida, M. Munoz-Ortuno, R. Herraez-Hernandez, J. Verdu-Andres, C. Molins-Legua, and P. Campins Falco Nitrites and Nitrates, Adnan Aydin Phosphates in Aquatic Systems, Alexandre Matthiensen, Juliana Antunes Galvao, and Marilia Oetterer Cyanides, Asbestos, Metals, and Si-Compounds Cyanides, Leo M. L. Nollet Asbestos in Water, James S. Webber Heavy Metals, Major Metals, Trace Elements, Jorge E. Marcovecchio, Sandra E. Botte, Claudia E. Domini, and Ruben H. Freije Determination of Silicon and Silicates, Salah M. Sultan Organic Parameters Main Parameters and Assays Involved with the Organic Pollution of Water, Lorena Vidal, Claudia E. Domini, and Antonio Canals Determination of Organic Nitrogen in the Aquatic Environment, Juliana Antunes Galvao, Alexandre Matthiensen, and Marilia Oetterer Determination of Urea in Aquatic Samples, Juliana Antunes Galvao, Alexandre Matthiensen, and Marilia Oetterer Organic Acids, Mercedes Gallego Fernandez, Evaristo Ballesteros Tribaldo, and Beatriz Jurado Sanchez Determination of Volatile Organic Compounds in Water, Ivan P. Roman Falco and Marta Nogueroles Moya Phenolic and Humic Compounds Determination of Phenolic Compounds in Water, Leo M. L. Nollet Characterization of Humic Matter, Leo M. L. Nollet Residues of Pesticides Determination of Pesticides in Water, Evaristo Ballesteros Tribaldo Analysis of Herbicide and Fungicide Residues in Waters, N. Sridhara Chary, Maria Jose Gomez Ramos, and Amadeo R. Fernandez-Alba Residues of PCBs, PCDDs, PCDFS, and PAHs Analysis of PCBs in Waters, L. Bartolome, O. Zuloaga, and N. Etxebarria PCDDs and PCDFs, Luigi Turrio-Baldassarri, Paola Pettine, and Laura Achene Polynuclear Aromatic Hydrocarbons, Chimezie Anyakora Surfactants and Petroleum Hydrocarbon Analysis Surfactants, Eva Pocurull and Rosa Maria Marce Petroleum Hydrocarbon Analysis, Ivan P. Roman Falco EDCs and Residues of Plastics Endocrine-Disrupting Chemicals, Pharmaceuticals, and Personal Care Products, Dimitra A. Lambropoulou and Eleni Evgenidou Residues of Plastics, Dimitra A. Lambropoulou and Eleni Evgenidou Index

Handbook of Water Analysis

Publisher Summary The chapter focuses on mycological aspects of the genus Metarhizium . The genus includes several species, varieties within species, and individual isolates with broad ranges of physiological traits—including host range. The interactions of these fungi with their hosts, and the large literature on their use for pest control, largely define the scientific and popular concepts of Metarhizium spp. Fungi of the hyphomycete genus Metarhizium have been isolated from infected insects and soil. Although some isolates of these fungi have rather restricted host ranges, the group is better known for its ability to kill a wide spectrum of insects, including insects in at least seven orders. The common name for Metarhizium -induced disease is “green muscardine,” based on the encrustation of insect cadavers with green conidia. The rapid increase in research on Metarhizium , followed by sustained high scientific output, can be explained by several important worldwide attitude changes and the initiation of several promising Metarhizium -based pest-control and molecular-biology efforts.

Metarhizium spp., cosmopolitan insect-pathogenic fungi: mycological aspects.

The comparative survival of various fecal indicator bacteria and enteric pathogens was studied in a stable well water supply by using membrane chambers There was more variation in the 29 coliform cultures and they died more rapidly, as a group, than the 20 enterococcus cultures that were examined The comparative survival of the organisms tested follows: Aeromonas sp > the shigellae (Shigella flexneri, S sonnei, and S dysenteriae) > fecal streptococci > coliforms = some salmonellae (Salmonella enteritidis ser paratyphi A and D, S enteritidis ser typhimurium) > Streptococcus equinus > Vibrio cholerae > Salmonella typhi > Streptococcus bovis > Salmonella enteritidis ser paratyphi B S bovis had a more rapid die-off than did S equinus, but both had significantly shorter half-lives than the other streptococci The natural populations of indicator bacteria from human and elk fecal material declined similarly to the pure cultures tested, whereas the die-off of fecal streptococci exceeded the coliforms from bovine fecal material

Comparative survival of indicator bacteria and enteric pathogens in well water.

The problems associated with recovery of pure cultures of Escherichia coli and Streptococcus faecalis from stream environments were examined utilizing membrane filter chambers. It was observed that upon exposure to the aquatic environment a significant proportion of cells lost their ability to produce colonies on a selective medium, yet retained this capability on a nutritionally rich, nonselective medium. Discrepancies in colony-forming units between nonselective and selective media indicated that a substantial portion of bacterial cells may become physiologically injured due to the environmental stress imposed by the aquatic environment. The extent of injury was observed to vary considerably among the eight different stream environments, since the amount of injury was not uniform for all types of water environments examined. It was observed that the injury acquired by a population of E. coli, during exposure to the aquatic environment, could be rapidly repaired in a nutritionally rich, nonselective medium. As the injured population of cells was exposed to the rich, nonselective broth, increasing proportions of cells were able to repair themselves such that they became insensitive to inhibitory agents in selective media.

Influence of Environmental Stress on Enumeration of Indicator Bacteria from Natural Waters

Various recovery methods used to detect coliforms in water were evaluated by applying the membrane filter chamber technique. The membrane filter chambers, containing pure-culture suspensions of Escherichia coli or natural suspensions of raw sewage, were immersed in the stream environment. Samples were withdrawn from the chamber at regular time intervals and enumerated by several detection methods. In general, multiple-tube fermentation techniques gave better recovery than plating or membrane filtration procedures. The least efficient method of recovery resulted when using membrane filtration procedures, especially as the exposure period of the organisms to the stream environment increased. A 2-h enrichment on a rich, nonselective medium before exposure to selective media improved the recovery of fecal coliforms with membrane filtration techniques. Substantially enhanced recoveries of E. coli from pure-culture suspensions and of fecal coliforms from raw-sewage suspensions were observed when compared with recoveries obtained by direct primary exposure to selective media. Such an enrichment period appears to provide a nontoxic environment for the gradual adjustment and repair of injured cells.

Evaluation of Recovery Methods to Detect Coliforms in Water

Bacteriological studies in 1968 and 1969 corroborated earlier findings that a municipal watershed which had been closed to public entry since 1917 yielded water with four to six times the coliform count found in an adjacent mountain watershed open to recreational activities. Similarly, chemical investigations showed higher concentrations of most ions in water from the closed area. Physiological differentiation of coliform and enterococcal bacteria revealed similar types of organisms in both animal droppings and stream water, with fecal coliforms accounting for as much as 70% of the coliform counts observed in the closed area in 1969. Opening of the closed drainage for limited recreation and expanded logging operations in the spring of 1970 coincided with an unexpected decrease in bacterial contamination of that stream. It is postulated that these human activities drove from the watershed a large wild animal population which had contributed substantially to the previous bacterial pollution. It would seem that the practice of closing high-mountain watersheds to public entry is questionable if governmental standards for water quality are to be met, and it also seems that the standards themselves should be reexamined.

G. K. Bissonnette

Papers

Comparative survival of indicator bacteria and enteric pathogens in well water.

Influence of Environmental Stress on Enumeration of Indicator Bacteria from Natural Waters

Evaluation of Recovery Methods to Detect Coliforms in Water

Effects of multiple use on water quality of high-mountain watersheds: bacteriological investigations of mountain streams.

Influence ofEnvironmental Stress on Enumeration ofIndicator Bacteria fromNatural Waters