Showing papers in "Methods in Microbiology in 2001"

Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes

Abstract: Publisher Summary The chapter discusses the fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes, and describes probe designing and testing. Fluorescence FISH with rRNA-targeted probes is a staining technique that allows phylogenetic identification of bacteria in mixed assemblages without prior cultivation by means of epifluorescence and confocal laser scanning microscopy, or by flow cytometry. FISH with oligonucleotide probes is for the purpose of bacterial identification that is to analyze bacterial community structure, and to follow the spatial and temporal dynamics of individual microbial populations in their habitat. Numerous aspects and applications of this method are discussed. FISH is successfully applied in freshwater, coastal, and offshore marine planktonic habitats, and in coastal sediments. It is shown that the fraction of bacteria detectable by FISH corresponds well with the abundance of active cells as determined by microautoradiography in coastal marine bacterioplankton.

Denaturing gradient gel electrophoresis in marine microbial ecology

Abstract: Publisher Summary Genetic fingerprinting techniques are excellently suited to comparison of large numbers of samples. Genetic fingerprinting of microbial communities provides banding patterns or profiles that reflect the genetic diversity of the community. Denaturing gradient gel electrophoresis (DGGE) of PCRamplified gene fragments is one of the genetic fingerprinting techniques used in microbial ecology. In DGGE, similar-sized DNA fragments are separated in a gradient of DNA denaturants according to differences in sequence. A variant of DGGE, temperature gradient gel electrophoresis (TGGE), makes use of a temperature gradient to separate gene fragments. DGGE is relatively easy to perform and is especially suited to the analysis of multiple samples. Since its introduction into microbial ecology, it has been adapted in many laboratories as a convenient tool for the assessment of microbial diversity in natural samples. It has been shown by several studies that the approach is reproducible and sensitive. Other new fingerprinting techniques, such as automated T-RFLP might be more sensitive but identification of predominant community members still requires cloning and sequencing of PCR products. A potential future development in PCR-DGGE fingerprinting might be to use fluorescently labelled PCR primers, which might (1) make staining of gels unnecessary, and (2) make it possible to add intra-lane standards with a different fluorochrome, facilitating gel-to-gel comparisons.

Measuring bacterial biomass production and growth rates from leucine incorporation in natural aquatic environments

Abstract: Publisher Summary An estimate of microbial production is used as a general index of microbial activity, and specifically, to calculate growth rates. Biomass production is used to obtain a first-order estimate of rates of several processes mediated by microbes. In the case of heterotrophic bacteria, biomass production is used to estimate use of dissolved organic material (DOM) if coupled with an estimate of the growth efficiency. DOM uptake equals bacterial biomass production divided by the growth efficiency (expressed as a fraction, not a percentage). Biomass production is the increase in biomass per unit time per unit volume or per area and is a function of both biomasses (B), usually expressed as carbon mass per volume—for example, μgC l-1—and the specific growth rate (μ) (e.g.h1). The methods used to examine bacterial production in aquatic environments are either thymidine (TdR) incorporation, or leucine (Leu) incorporation. Both are rapid, easy, and specific for heterotrophic bacteria. This leucine method is discussed, as it is more straightforward than the TdR method for estimating the bacterial production. Two variations of the Leu method are described; however, the basic biochemistry and physiology behind the methods are the same.

Nitrogen fixation: Nitrogenase genes and gene expression

Abstract: Publisher Summary This chapter discusses nitrogenase genes and gene expressions. The primers used for nifH amplification, the methods used to extract genomic DNA and mRNA, the alignment and analysis of nifH sequences, and the RT-PCR protocol are described. Biological nitrogen fixation is the enzymatic reduction of atmospheric dinitrogen to ammonium. The conventional nitrogenase enzyme is encoded by the nifHDK genes, which are in contiguous arrangement within the genome. Alternative nitrogenases (alternative and second alternative) also contain nifH, but contain a third protein in the counterpart to the Mo protein, which is encoded by nifG (nifDGK). Nitrogenase genes can be detected and characterized by amplification from environmental samples using the polymerase chain reaction (PCR). Amplification of nitrogenase genes indicates that nitrogen-fixing microorganisms are present, but not whether or not they are actively fixing nitrogen. By coupling, the PCR assay with reverse transcription (RT-PCR) microorganisms that are actively expressing the nitrogenase enzyme can be detected. Once genes are amplified, the diversity of sequences can be determined by a number of means, including cloning and sequencing of individual amplification products.

Nitrogen fixation and denitrification

Abstract: Publisher Summary The chapter discusses nitrogen fixation, denitrification, and their applications. It discusses the most widely used enzyme-based and direct tracer procedures in the N cycle- N 2 fixation and denitrification. Biological nitrogen fixation is the enzymological capacity of certain bacteria and Archaea to convert gaseous dinitrogen to ammonium on a pathway to amino acid synthesis. In marine systems, the attention is focused on diazotrophic cyanobacteria, because of their quantitative importance in supplying new N to the upper ocean. Nitrogenase activity is routinely determined in many laboratories using the C 2 H 2 reduction procedure. Denitrification is measured conveniently in many microbial systems by addition of C 2 H 2 , which inhibits N 2 O reductase, the last step of the denitrification pathway. The presence of C 2 H 2 results in the accumulation of N 2 O, which is detected selectively and sensitively by electron capture gas chromatography. Many of the procedures and conceptual design of denitrification experiments parallel those of the C 2 H 2 reduction procedure. As for nitrogen fixation, denitrification can also be measured directly by the introduction of enriched 15-N nitrate (or nitrite) into a system, with the determination of progressive enrichment of 15N in the N 2 pool.

Enumeration of total and highly active bacteria

Abstract: Publisher Summary This chapter covers standard methods of enumeration of total bacterial numbers with epifluorescence microscopy. The advantages of flow cytometery over microscopic enumeration are discussed. Conversion factors used to estimate carbon biomass based on cell abundance and cell sizes are discussed, and also the Circulating Tumor Cell (CTC) assay, which identifies bacteria with highly active electron transport systems (ETS) are described. Bacterial abundance, cell size, biomass, and activity are among the most fundamental properties that characterize natural aquatic systems, and the measurement of these parameters is central to aquatic microbial ecology. Epifluorescence techiques are now routinely performed in most microbial laboratories, and image analysis has increasingly been used to determine cell size and other cellular properties. Flow cytometry offers the possibility of assessing not only total abundance with great speed and precision, but also properties of individual cells, such as size and metabolic activity. Cytometry also offers the possibility of physically separating, via cell sorting, subpopulations based on various cellular properties. Flow cytometry and image analysis combined with selective staining methods, permit determination of physiological condition of in situ bacterioplankton.

Flow cytometric analysis of autotrophic picoplankton

Abstract: Publisher Summary The chapter discusses the principles of flow cytometry and important methodology and applications. It discusses the analysis of autotrophic picoplankton using a popular benchtop instrument, the FACSCalibur (Becton Dickinson). Flow cytometry is the analysis of light scatter and fluorescence emitted from individual cells as they flow individually though an intensely focused light source at a rate of thousands of cells per second. In the detection system, the light pulse is converted to an electrical signal (voltage) by either a photomultiplier (PMT) or photodiode. Flow cytometry has become an indispensable tool in marine microbial ecology for the identification and enumeration of picoplankton, that is, cells that are

Fingerprinting viral assemblages by Pulsed Field Gel Electrophoresis (PFGE)

Abstract: Publisher Summary Viruses are the most abundant microorganisms in marine and freshwater environments, and perhaps the most genetically diverse. DNA-based fingerprinting approaches, which rely on amplification of rRNA gene fragments by polymerase chain reaction (PCR), have facilitated analyses of bacterial community composition. These approaches have a more restricted application when analyzing viral assemblages, because of the extreme genetic diversity among viruses. Unlike bacteria, there are no gene sequences conserved in all viruses, which can serve as universal primer sites for PCR amplification. The approach described here uses variation in genome size as the basis for obtaining a fingerprint of a viral assemblage. A whole genome fingerprinting approach is possible, because viral genomes can vary greatly in length (a few thousand to hundreds of thousands of base pairs) yet they fall within a range that is easily resolved using pulsed field gel electrophoresis (PFGE). The PFGE fingerprinting technique provides a quick and relatively simple means of visualizing differences in the composition of viral assemblages.

Phosphorus cycle in seawater: Dissolved and particulate pool inventories and selected phosphorus fluxes

Abstract: Publisher Summary The chapter presents the principle and stepwise procedures for the accurate estimation of (1) dissolved orthophosphate (HPO42; hereafter referred to as “Pi”), soluble reactive P (SRP), total dissolved P (TDP) and particulate P (PP) as minimal constraints on P pool inventories; (2) dissolved and particulate adenosine-5'-triphosphate concentrations (D-ATP and P-ATP, respectively); (3) Pi uptake/regeneration and dissolved organic P (DOP) production rates using radiolabeled Pi (32P or 33P) precursors; and (4) turnover rate estimation of the α-P and γ-P of intracellular ATP. The latter measurements provide information on microbial growth rate, total energy flux and the biologically available P (BAP) pool. These methods collectively provide a protocol suite that is suitable for an initial study of P dynamics in selected marine habitats.

Protistan herbivory and bacterivory

Abstract: Publisher Summary This chapter describes the two experimental protocols commonly used to examine bacterivory and herbivory by phagotrophic protists. Preceding the description of each method is a short discussion of the theory, advantages, and disadvantages of experimental approaches for measuring protistan food web interactions. A number of variations on these methods and alternative approaches are discussed in this chapter. Reference to alternative protocols and variations on these two methods are provided as space permits. Experimental approaches for measuring protistan trophic activity—that is, herbivory and bacterivory—is characterized according to the taxonomic level at which they are directed, and the general approach taken to assess the activity of the assemblage. Experimental designs are classified into one of two general approaches; (1) perturbation of trophic relationships and monitoring consequent changes in prey abundance/biomass, or (2) tracer experiments that attempt to follow the uptake of prey into consumers or their removal from the water.

Enumeration of viruses

Abstract: 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.

Lysogeny and transduction

Abstract: Publisher Summary Lysogeny and transduction describes a type of phage/host interaction and a method of bacterial gene transfer (procaryotic sex), respectively. This chapter describes methods that have been found useful in studying lysogeny and transduction in the marine environment. Lysogeny occurs when a phage enters into a stable symbiosis with its host. The host (bacterium or algal cell) and phage capable of entering into such a relationship are termed a “lysogen and temperate phage,” respectively. The temperate phage genome becomes integrated into one of the replicons of the cell (chromosome, plasmid, or another temperate phage genome) and is termed a “prophage.” In transduction, the genes are originated in a bacterial host and are not a normal part of the phage genome. The detection of lysogeny in cultures or natural populations is usually through prophage induction by use of a mutagenic agent, usually mitomycin C. The methods described are all based on some derivative of this procedure. Bacteriophage-mediated transduction is one of three well-known mechanisms, along with conjugation and transformation, of horizontal gene transfer among prokaryotic organisms. In transduction, bacterial DNA or plasmid DNA is encapsulated into phage particles during lytic replication of the phage in the donor cell and is transferred to the recipient cell by infection. This donor DNA either undergoes recombination with the host chromosome to produce a stable transductant or remains extrachromosomal as a plasmid.

Fungal biomass and productivity

Abstract: Publisher Summary This chapter discusses fungal biomass and productivity. Fungal masses are best measured via biochemical indices because fungi digest natural materials such as saltmarsh grass from within the opaque solid substrate. Fungi (including chytrids) and oomycotes are part of the marine microbiota; however, biochemical-index methods are developed for biomass and productivity measurement with natural samples only for true fungi. Biomasses of zoosporic, non-mycelial true fungi (the chytrids) is measured using hexosamine techniques, but not with the ergosterol technique because chytrids do not synthesize ergosterol. It is possible to measure fungal biomass via biochemical proxies in two very different ways: as total mass (living plus dead, evacuated hyphae) or as living mass (membrane-containing mycelium). The hexosamine methods yield total-mass values (as chitin of cell walls is not readily lysed upon hyphal death), and the ergosterol methods yield living-mass values (as ergosterol is a membrane component, and membranes are readily lysed upon hyphal death). The method for finding living-fungal mass and fungal productivity is described in this chapter.

Human enteric viruses and parasites in the marine environment

Abstract: Publisher Summary Water is a common vehicle for the transmission of many enteric viruses. Enteric viruses survive longer in fresh and marine water than coliform bacteria, which are used to monitor water quality. In marine and other surface waters, numbers of enteric viruses are often too low to be detected in unconcentrated samples. Therefore, large volumes of water must be concentrated by adsorption/filtration and elution (desorption) before analysis. More than 100 types of human pathogenic viruses are present in fecally contaminated water, and are detected by the current available methods. The detection of adenoviruses, hepatitis A virus, astroviruses, and rotaviruses by cell culture is possible; however, the methods are tedious and less often used. Nucleic acid hybridization and the polymerase chain reaction (PCR) are used for detecting human viruses and other fastidious microbial pathogens in the environment. Molecular detection methods are important in studying the occurrence of Hepatitis A and Norwalk-like viruses, which are epidemiologically important pathogens but do not produce cytopathogenic effects (CPE) readily in cell culture. Molecular techniques have also enhanced the speed and sensitivity of detection for the more routinely cultured enteroviruses.

Estimating viral proliferation in aquatic samples

Abstract: 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.

Methods for psychrophilic bacteria

Abstract: Publisher Summary This chapter discusses the various procedures useful for the isolation and study of a specific group of bacteria, the psychrophiles. Most procedures detailed are general methods modified for cold-adapted bacteria, but are broadly applicable in the study of marine bacteria. These bacteria have temperature optima at 20°C or more, but are able to grow at 0°C. The growth rates of psychrotolerant bacteria are usually equivalent to or better than that of psychrophiles at low temperatures. Psychrotolerant bacteria abound even in the coldest of environments, simply because many of them are ecophysiologically resilient and nutritionally versatile species. The chapter first covers isolation, routine cultivation, and maintenance of psychrophiles. Procedures for determining cardinal growth temperatures, using temperature gradient incubator (TGI), are then detailed. Techniques for definitively identifying and quantifying fatty acids implicated in cold adaptation of cellular membranes including polyunsaturated fatty acids (PUFA) and branched chain fatty acids are explained. Phenotypic characterization for identification and taxonomic purposes is also covered including a list of phenotypic tests applicable to studying marine psychrophiles and marine bacteria in general.

Collection and identification of marine yeasts

Abstract: Publisher Summary This chapter discusses methods of isolation and collection of marine yeasts using traditional techniques, and identification of species and strains with molecular methods. Traditional grabs, gravity, sterile bag samplers, and piston cores, or submersibles are used for collection of yeast from deep-water sediments. Traditional methods of yeast isolation and cultivation are employed for obtaining large numbers of isolates. Classical techniques for the identification of species of yeasts require morphological, physiological, and biochemical tests. Species can be identified by sequence analysis of purified isolates or via species-specific primers and hybridization probes from purified isolates or from field samples of mixed species. Differentiation of strains within a species plays a significant role in ecological population analyses. The intergenic spacer region (IGS) is studied for population and strain level differences among a wide range of eukaryotes, including plants, invertebrates, and yeasts. The polymerase chain reaction (PCR) detection technique is useful for identification of small numbers of species; however, when several species need to be identified, the PCR matrix is difficult to handle. Therefore, techniques that are more appropriate will include hybridization probes with macro- and microarrays designed to identify large numbers of species.

Deep-sea piezophilic bacteria

Abstract: Publisher Summary The chapter discusses deep-sea piezophillic bacteria. Hyperpiezopsychrophiles are psychrophilic microorganisms having a maximum growth rate at a pressure greater than 50 MPa and grow poorly, if at all, at atmospheric pressure. They are the most pressure adapted bacteria known. Their cultivation is possible starting with unwarmed, decompressed samples even of the greatest ocean depths (beyond 10 500 m) if the enrichment cultures are compressed within an hour or so of the sample's arrival on board ship. Hyperpiezopsychrophiles from a 6000 m depth survive in water samples kept on ice at atmospheric pressure for two weeks. High-pressure laboratory methods are indispensable for pure-culture deep-sea microbiology. Enrichment cultures incubated at the pressure and temperature of that depth yield true inhabitants of that depth. The laboratory methods described in this chapter are useful for the enrichment, isolation, and pure culture study of some of the microbial inhabitants of deep-sea and subsurface habitats. The methods are accessible to anyone wishing to make a modest investment in high-pressure equipment.

Microbial ecology at sea: Sampling, subsampling and incubation considerations

Abstract: Publisher Summary This chapter provides a few general guidelines on sampling, subsampling, and other relevant field experimental design criteria on marine microorganisms. Many distinct marine ecosystems and their microbial assemblages are identified and studied, ranging from ice-swept polar seas to deep-sea hydrothermal vents. Sampling is one of the most important, but often overlooked, aspects of oceanography. The successful isolation of deep-sea bacterial isolates that are obligately barophilic has emphasized that pressure is an important determinant of microbiological zonation in the sea. Many techniques currently employed in microbiological oceanography require incubation of a seawater sample for various periods of time. The underlying assumption of these methods is that the subsequent incubation conditions do not alter the in situ rates of metabolism or biosynthesis. An equally important concern, especially for post-collection incubation measurements, is attention to a clean sampling technique. Metal samplers, toxic closure components or other potentially detrimental materials should be avoided. In order to ensure that the rates measured during the post-collection incubation procedure are representative of those occurring in nature, several precautions must be taken.

Determining the physiological status of individual bacterial cells

Abstract: Publisher Summary This chapter determines the physiological status of individual bacterial cells. are used to estimate the physiological status of individual cells in marine and estuarine water samples. (1)The Vital Stain and Probe (VSP) method is an epifluorescent direct microscopy counting technique that combines the general DNA stain DAPI, the vital stain propidium iodide (PI), and 16S rRNA targeted oligonucleotide probes to identify bacterial cells in situ . (2) Nucleoid staining technique in which the nucleoid is observed with epifluorescence microscopy when it is stained with a DNA fluorochrome such as DAPI. (3) Microautoradiography is a technique that detects the cellular localization of a radiolabeled substance ill situ. This method combines microautoradiography with 16S ribosomal RNA-targeted oligonucleotide probes, which allows simultaneous in situ identification and determination of substrate uptake patterns of individual cells. (4) Live/dead baclight tm bacterial viability kit provides a two-color fluorescent assay of bacterial viability. The kit utilizes mixtures of two nucleic acid stains: SYTO 9 and Propidium Iodide (PI). It is a membrane-impermeant stain that can passively diffuse through a cell wall and act as an indicator of loss in membrane integrity and thus as an indicator of cell viability. (5) Direct viable counting [DVC]: Kogure method utilizes the antibiotic nalidixic acid to interfere with normal cell division. In the presence of this antibiotic, sensitive cells grow but do not divide and can therefore be easily identified microscopically as elongated or enlarged cells.

Quantification of algal viruses in marine samples

Abstract: Publisher Summary This chapter describes the current methods available for the enumeration of specific viruses infecting phytoplankton in aquatic environments. It represents a compilation of methods that are employed in classic virology, and those that are adapted for use by viral ecologists working in natural systems. Two approaches, the plaque assay and most probable number (MPN) assay, are described, which allow researchers to both enumerate and isolate viruses that lytically infect marine photoautotrophs. Using a dilution approach, abundance of viruses in a sample can be estimated. This process is based on the theoretical assumption that a single infectious virus can destroy a population of sensitive host cells (given time). The MPN approach to quantifying infectious viruses involves the exposure of a series of log-based dilutions of the sample containing the viruses to a liquid culture of host cells. One problem commonly associated with the screening of natural samples is the breakthrough of unwanted organisms—for example, bacteria and protozoans—through the filter into the sample to be screened. Plaque assays are commonly used in bacteriophage studies in order to enumerate the abundance of infectious phage in a sample. These same techniques are applied to the enumeration of phycoviruses..

Molecular phylogeny: Applications and implications for marine microbiology

Abstract: Publisher Summary This chapter describes the application of modern molecular biological techniques to marine microbiology. Promising molecular based applications are also viable alternatives such as fluorescent in situ hydridization (FISH) of group-specific oligonucleotide probes or the high-throughput method of terminal restriction fragment length polymorphism (T-RFLP) used to track specific populations through space and time. Numerous studies using these molecular biological approaches have significantly changed the understanding of marine microbiology, fueling new avenues of research—for example, (1) the initial dissections of bacterioplankton communities in the Atlantic and Pacific Oceans, (2) the discovery of archaeoplankton, and (3) the discovery of dominant populations of iron- and sulfur-oxidizing bacteria at hydrothermal vents. This approach is used in marine microbiology to apply phylogenetic analysis to establish evolutionary relationships among organisms, and to use this information as a framework for making inferences about community structure, genetic and thereby inferred organismal diversity, and (to a lesser degree) to infer physiological adaptation when applicable. The experimental design based on molecular biological techniques can yield information regarding both autecology and synecology, in terms of community structure and phylogenetic diversity, and is analogous to taking a census of a community and estimating a roadmap of evolutionary relationships for individual populations contained within.

Introduction, history, and overview: The ‘methods’ to our madness

Abstract: Publisher Summary The chapter discusses the relationship between methods and conceptual advances in marine microbiology Revolutionary discoveries in marine microbiology during the past two decades have fundamentally changed the concepts of the structure and functioning of marine ecosystems Early microbial oceanographers used the viable count and pure culture technique to determine the abundance, distribution, and species composition of bacteria The culture technique was not enough, and a direct and quantitative ecosystem analysis was required Then the emphasis on culture-independent methods to study natural microbial assemblages led to a flurry of biochemical methods for measuring bacterial (and total microbial) biomass and the physiological state of natural assemblages of marine microbes: ATP for total microbial biomass; lipopolysaccharide and muramic for bacterial biomass; electron transport system (ETS) activity as a proxy for microbial respiration; energy charge as a measure of the physiological state of the assemblage (for instance in response to ecosystem stress) Method refinement to measure respiration of natural bacterial or protozoan contributes to understanding microbial carbon cycling in the ocean One of most influential techniques in microbial oceanography is Hans- Georg Hoppe's method for measuring bacterial ectohydrolase activities in intact planktonic It has provided important insights and constraints on how the organic matter in particles and polymers become available for uptake by bacteria

Radiotracer assays (35S) of sulfate reduction rates in marine and freshwater sediments

Abstract: Publisher Summary The current understanding of sulfate reduction has been derived from assays based on 35 SO 4 2 , a readily available and relatively inexpensive radiotracer that can be purchased in a carrier-free form with very high specific activity. Prior to routine use of radiolabeled sulfate, sulfate reduction rates in marine sediments were assayed successfully with depletion methods and diagenetic modeling. Depletion approaches, which simply measure loss of sulfate over time, have proven suitable in cases where extended incubations are feasible. Depletion approaches, which have no significant regulatory concerns—for example, disposal of radioactive wastes—, and which require minimal sample processing and analytical facilities, have offered attractive nonisotopic alternatives to radioassays. Diagenetic modeling, which is based on the use of diffusion-reaction models and sulfate and organic matter concentrations, has been particularly useful for estimating sulfate reduction in deep-sea sediments, where activity occurs at low rates over many meters of the sediment column. Advantages of 35 SO 4 2 include a high level of sensitivity, even for very low rates of activity, the possibility of assessing activity in the presence of sulfide oxidation, short incubation times under near in situ conditions and information on end product formation—for example, elemental sulfur, pyrite.

Optical remote sensing techniques in biological oceanography

Abstract: Publisher Summary This chapter describes the basics of ocean color remote sensing. It includes a description to obtain and use SeaWiFS data within NASA's freely available ocean color remote sensing software. Differences in methodology and some of the more recent developments in the optical remote sensing field are described. By exploring how light penetrates the water column and how the optical constituents affects the light as it travels through the water, the basic understanding of the value and limitations of ocean color data is provided. Remote sensing provides a tool that can provide information over time/space scales not possible using traditional sampling approaches from ships. SeaWiFS data can be acquired from NASA's Distributed Active Archive Center [DAAC], and can be ordered online. SeaWiFS is a commercial instrument flying on Orbimage's Orbview-2 spacecraft. Apart from the other image processing packages available, the chapter describes SeaDAS, as it is free and can be used with a currently operational satellite sensor (SeaWiFS). There are many other ocean color satellites being planned (and one that has just been launched), but the data streams are not currently available.

In situ PCR/RT-PCR coupled with in situ hybridization for detection of functional gene and gene expression in prokaryotic cells

Abstract: Publisher Summary This chapter discusses the detailed protocols for performing in situ PCR (PI-PCR) on microscope slides, including prokaryotic in situ polymerase chain reaction (PCR), in situ RT-PCR, and in situ hybridization. The methods are used to detect the functional gene and gene expression inside bacterial cells of mixed culture, enriched microcosms, and natural communities. PCR technology is used to amplify and detect the presence of several functional genes in aquatic microbial communities. In vitro PCR/RT-PCR performed on bulk extraction of nucleic acids is useful for detecting the presence of genes that are associated with a specific function of a microbial community. In situ PCR is used to specifically amplify and detect single copy or low copy nucleic acid sequences in single cells and tissue sections. PI-PCR and PI-RT-PCR are coupled with flow cytometry to enumerate bacterial cells with a specific functional gene and gene expression. PI-RT-PCR with labeled primers demonstrated the potential to detect poorly expressed mRNA in individual bacterial cells, and this approach is used to detect the expression of several functional genes in individual Salmonella typhimurium cells. PI-PCR coupled with FISH has been found to provide a higher specificity than other protocols. The combination of PI-PCR and FISH reduces the false positive signal as the initial in situ PCR/RT-PCR is performed with nonlabeled nucleotides. The stringency of FISH can be critically optimized by varying hybridization conditions—e.g. temperature, formamide and salt—to achieve more specific detection with low background.

Methods for the study of hydrothermal vent microbes

Abstract: Publisher Summary The chapter discusses various ways in which microbiological samples can be obtained from deep-sea vents, and how these samples are processed for DNA extraction or enrichment culturing. The most challenging aspects of doing deep-sea microbiological research are obtaining a representative and uncontaminated sample. The types of samples that can be collected include high-temperature fluids, diffuse flow (low-temperature, shimmering water) water samples, microbial mats, rock and sulfide mineral samples, epibionts and symbionts associated with animals. Deep-sea hydrothermal vents have been explored to depths of over 3700 m. Pressure, therefore, is a consideration when exploring a microbial sampling strategy. Several laboratories have designed microbial sampling devices that maintain the in situ pressure. A description of one such device that is also an in situ growth experiment is described in this chapter. Two isolates, Thermococcus barophilus and Palaeococcus ferrophilus appear to be moderate barophiles or piezophiles. There will be an increasing need to develop in situ technologies to measure microbial activity and diversity at deep-sea hydrothermal vents..

Isolation of oligobacteria

Abstract: Publisher Summary The chapter discusses the methods for isolation of oligobacteria. Oligobacteria are defined as bacteria that can accumulate dissolved organics from low concentrations. Oligobacteria are smaller than copeotrophs giving rise to the term ultramicrobacteria. They are low in solids content and DNA probably as a way of maximizing use of their energy reserves for growth and increasing their surface to volume ratio to maximize their specific affinities for nutrients. There are two methods of isolation discussed: (1) spread plate isolation and (2) extinction culture isolation. The procedures outlined are seen as a significant improvement over conventional techniques for isolating and thus understanding pelagic aquatic bacteria. The frequency of isolation success has been increased by use of extinction cultures where viabilities can approach 10% even in oligotrophic low-productivity waters. The extinction culture technique is based on the observation that when bacteria are filtered from seawater and then re-inoculated into filtered seawater, the population will regenerate. The resulting populations were first called “seawater cultures.” When the inoculum is large—that is, 10% unfiltered seawater—the rate of increase can be used to determine the rate of growth. When it is small, just a few organisms, it can be used in combination with the number of organisms inoculated to determine the culturability or viability of those originally present.

Measurement of UVB-induced DNA damage in marine planktonic communities

Abstract: Publisher Summary Ultraviolet radiation (UVR) has been recognized for many years as a potential stress for organisms in a variety of environments. Decrease in phytoplankton production may result in a decline in bacterial production, which may be compounded by direct UVB effects on bacterioplankton. UVR may directly affect viruses, bacteria, phytoplankton, or zooplankton via direct DNA damage and reduced rates of production. The effects of UVR on marine bacterioplankton are investigated by using radiolabeled precursor molecules such as 3 H thymidine, and 3 H- or 14 C-leucine. It has been shown that bacterioplankton experience significant amounts of DNA damage (CPDs) in surface waters, often twice the amount of larger eukaryotic cells. Bacterioplankton accumulate DNA damage over a solar day. DNA damage extend to depths of 10 m or more in calm waters but the amount of damage may be significantly altered by surface water mixing events. Radioimmunoassay (RIA) technique is used for the measurement of specific DNA photoproducts in the DNA of UV-irradiated cells. DNA damage results are reported per unit (megabase) DNA, and are therefore independent of the concentration of DNA present in the original sample or the amount of DNA assayed. The sensitivity of the RIA is determined by the affinity of the antibody and specific activity of the radiolabeled antigen (probe).

Gene expression by mRNA analysis

Abstract: Publisher Summary Gene expression studies are very important in the study of marine heterotrophic bacteria, particularly the extremophiles, in terms of understanding the response to pressure and low and high temperature. The capability to detect gene expression by mRNA analysis has enabled the real-time detection of expression of genes involved in bioremediation as well. Gene expression encompasses transcription, translation, post-translational modification, and production of a functional gene product. This chapter describes three related methods for mRNA isolation and compares some results obtained by each. Two RNA extraction methods; Guanidinium isothiocyanate phenol sarcosyl (GIPS) method and Boom methods are discussed. Dotting, probing, quantitating mRNA levels, and application of assay are also described. Extraction of natural populations of phytoplankton from Ft. Desoto, St Petersburg, FL and probed with the Form ID probe indicated that the Boom protocol resulted in hybridization that was at least as great as that with the GIPS reagent.