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Nucleic acid-based analytical methods, ranging from species-targeted PCRs to metagenomics, have greatly expanded our understanding of microbiological diversity in natural samples. However, these methods provide only limited information on the activities and physiological states of microorganisms in samples. Even the most fundamental physiological state, viability, cannot be assessed cross-sectionally by standard DNA-targeted methods such as PCR. New PCR-based strategies, collectively called molecular viability analyses, have been developed that differentiate nucleic acids associated with viable cells from those associated with inactivated cells. In order to maximize the utility of these methods and to correctly interpret results, it is necessary to consider the physiological diversity of life and death in the microbial world. This article reviews molecular viability analysis in that context and discusses future opportunities for these strategies in genetic, metagenomic, and single-cell microbiology.Yet it hath happened that the veritable body without the spirit hath walked.-Ambrose Bierce, The Death of Halpin Frayser M icrobiologists, like characters in zombie fiction, quickly learn the critical importance of distinguishing the living from the dead. In addition to characterizing the numbers and species of microorganisms in samples, it is important to collect data on their physiological states. The most fundamental physiological state of microbial cells is their viability, defined here as the capacity to form progeny. For ecologists, pathobiologists, metagenomicists, food or water safety analysts, infectious-disease clinicians, and virtually every other stripe of microbiologist, the observation of a viable microorganism in a sample means something entirely different from the observation of a dead one. Despite its importance, this distinction remains extremely challenging by current microbiological methods.Microbiological culture meets this requirement, as it selectively detects viable organisms. However, because only a small percentage of species can be cultured, this strategy underestimates microbial diversity (1-6). In contrast, nucleic acid-based methods, ranging from species-specific PCR to metagenomic methods, have greatly advanced our ability to detect diverse microorganisms independently of microbiological culture (7,8). However, these methods provide only limited information on the physiology of microorganisms in samples. They can assess microbial viability retrospectively, by measuring quantitative changes over time, but they cannot discern viability cross-sectionally (in single measurements). Traditional PCR is notoriously poor at differentiating DNA associated with a viable bacterial cell from DNA associated with an inactivated one or from a free DNA fragment. All of these analytes register as "hits" in PCR, despite their very distinct meanings.In order to address this limitation, alternative PCR-based strategies have been developed. This article reviews two complementary strategies. One strategy, term...

Innovative research relating oceans and human health is advancing our understanding of diseasecausing organisms in coastal ecosystems. Novel techniques are elucidating the loading, transport and fate of pathogens in coastal ecosystems, and identifying sources of contamination. This research is facilitating improved risk assessments for seafood consumers and those who use the oceans for recreation. A number of challenges still remain and define future directions of research and public policy. Sample processing and molecular detection techniques need to be advanced to allow rapid and specific identification of microbes of public health concern from complex environmental samples. Water quality standards need to be updated to more accurately reflect health risks and to provide managers with improved tools for decision-making. Greater discrimination of virulent versus harmless microbes is needed to identify environmental reservoirs of pathogens and factors leading to human infections. Investigations must include examination of microbial community dynamics that may be important from a human health perspective. Further research is needed to evaluate the ecology of non-enteric water-transmitted diseases. Sentinels should also be established and monitored, providing early warning of dangers to ecosystem health. Taken together, this effort will provide more reliable information about public health risks associated with beaches and seafood consumption, and how human activities can affect their exposure to diseasecausing organisms from the oceans.

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