Rachel T. Noble

Rapid Virus Production and Removal as Measured with Fluorescently Labeled Viruses as Tracers

ABSTRACT Pelagic marine viruses have been shown to cause significant mortality of heterotrophic bacteria, cyanobacteria, and phytoplankton. It was previously demonstrated, in nearshore California waters, that viruses contributed to up to 50% of bacterial mortality, comparable to protists. However, in less productive waters, rates of virus production and removal and estimates of virus-mediated bacterial mortality have been difficult to determine. We have measured rates of virus production and removal, in nearshore and offshore California waters, by using fluorescently labeled viruses (FLV) as tracers. Our approach is mathematically similar to the isotope dilution technique, employed in the past to simultaneously measure the release and uptake of ammonia and amino acids. The results indicated overall virus removal rates in the dark ranging from 1.8 to 6.2% h−1 and production rates in the dark ranging from 1.9 to 6.1% h−1, corresponding to turnover times of virus populations of 1 to 2 days, even in oligotrophic offshore waters. Virus removal rates determined by the FLV tracer method were compared to rates of virus degradation, determined at the same locations by radiolabeling methods, and were similar even though the current FLV method is suitable for only dark incubations. Our results support previous findings that virus impacts on bacterial populations may be more important in some environments and less so in others. This new method can be used to determine rates of virus degradation, production, and turnover in eutrophic, mesotrophic, and oligotrophic waters and will provide important inputs for future investigations of microbial food webs.

A Regional Survey of the Microbiological Water Quality Along The Shoreline Of The Southern California Bight

A regional survey of the microbiological water quality along the shoreline of the Southern California Bight (SCB), from Point Conception south to Ensenada, Mexico, was conducted during August, 1998, by 36 agencies under the coordination of the Southern California Coastal Water Research Project (SCCWRP). Microbiological water quality was assessed by calculating the percentage of shoreline-mile-days that exceeded bacterial indicator thresholds for total and fecal coliforms, total/fecal ratios, and enterococci. Sample sites were selected using a stratified random sampling approach, with the SCB recreational shoreline divided into six strata: high- and low-use sandy beaches, high- and low-use rocky shoreline, and perennial and ephemeral freshwater outlets. Samples were collected on a weekly basis at a total of 253 sites, beginning on August 2nd, 1998 and continuing for five weeks. Samples were analyzed by 22 participating labs using their normal methods (multiple tube fermentation, membrane filtration, Colilert® and/or Enterolert®). All labs met testing criteria established through intercalibration exercises and quality control check samples distributed during the sampling period. Nearly 95% of the shoreline-mile days did not exceed daily and monthly bacterial indicator thresholds, demonstrating good bacteriological water quality along the SCB shoreline. Freshwater outlets, comprised mainly of storm drains, had the poorest water quality with 60% and 40% of the shoreline-miles exceeding monthly and daily thresholds, respectively. Freshwater outlets were also more likely to demonstrate exceedances by multiple indicators at a single site, and repeat exceedances at sites over the five-week period. Compared with the southern California beaches, Mexican beaches had nearly 5 times the number of exceedances for total and fecal coliforms, and nearly 8 times the number of exceedances for total/fecal ratios.

Enumeration of viruses

Publisher Summary This chapter provides the details necessary for the enumeration of viruses and bacteria in seawater using the nucleic acid stain SYBR Green I. Current research in the fields of marine microbiology and marine microbial ecology requires the ability to enumerate viruses and bacteria. In the past, counting microbes in seawater samples by transmission electron microscopy (TEM) was the standard method. SYBR Green I stain was originally used for research using flow cytometry. SYBR Green I is a viable tool, which yields virus counts comparable to TEM in a broad variety of samples, and seems to be more easily applied to the analysis of seawater samples than some of the previously mentioned stains. SYBR Green I has the advantage of being usable in conjunction with seawater and commonly used fixatives and a short staining period. SYBR Green I stained viruses and bacteria are intensely stained and easy to distinguish from other particles with both older and newer generation epifluorescence microscopes. SYBR Green I is inexpensive and is less carcinogenic than other typical nucleic acid stains. It has recently been noted that another nucleic acid stain, SYBR Gold, is used interchangeably with SYBR Green I. This stain appears to require a slightly shorter staining time (12 min), and is less expensive.

Estimating viral proliferation in aquatic samples

Publisher Summary Estimates of viral production and decay rates provided the valuable confirmation that viruses are active members of the marine community. Viral production involves the lysis of host cells and the release of cellular material as dissolved and colloidal organic carbon. Therefore, measurements of viral replication rates are also useful for assessing the contribution of viruses to bacterial mortality and organic matter cycling in the ocean. By assuming a burst size, viral productivity can be used to estimate rates of bacterial lysis. This approach provides an additional means to assess bacterial mortality along with the visualization of intracellular viral particles by transmission electron microscopy. A wide variety of different approaches for measuring viral productivity include: (1) quantifying net increases in viral abundance over time; (2) measuring rates of viral decay; (3) estimating viral DNA synthesis rates by radiolabeling; (4) calculating expected viral release rates from estimated rates of bacterial lysis and an assumed burst size; and (5) measuring tracer dilution rates using fluorescently labeled viruses (FLV) as tracers.