One of the prerequisites of making ecological conclusions derived from genetic fingerprints is that bacterial community profiles reflect the live portion of the sample of interest. Propidium monoazide is a membraneimpermeant dye that selectively penetrates cells with compromised membranes, which can be considered dead. Once inside the cells, PMA intercalates into the DNA and can be covalently cross-linked to it, which strongly inhibits PCR amplification. By using PCR after PMA treatment, the analysis of bacterial communities can theoretically be limited to cells with intact cell membranes. Four experiments were performed to study the usefulness of PMA treatment of mixed bacterial communities comprising both intact and compromised cells in combination with end-point PCR by generating community profiles from the following samples: (i) defined mixtures of live and isopropanol-killed cells from pure cultures of random environmental isolates, (ii) wastewater treatment plant influent spiked with defined ratios of live and dead cells, (iii) selected environmental communities, and (iv) a water sediment sample exposed to increasing heat stress. Regions of 16S rRNA genes were PCR amplified from extracted genomic DNA, and PCR products were analyzed by using denaturing gradient gel electrophoresis (DGGE). Results from the first two experiments show that PMA treatment can be of value with end-point PCR by suppressing amplification of DNA from killed cells. The last two experiments suggest that PMA treatment can affect banding patterns in DGGE community profiles and their intensities, although the intrinsic limitations of end-point PCR have to be taken into consideration.

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Microbial ecology has undergone a profound change in the last two decades with regard to methods employed for the analysis of natural communities. Emphasis has shifted from culturing to the analysis of signature molecules including molecular DNA-based approaches that rely either on direct cloning and sequencing of DNA fragments (shotgun cloning) or often rely on prior amplification of target sequences by use of the polymerase chain reaction (PCR). The pool of PCR products can again be either cloned and sequenced or can be subjected to an increasing variety of genetic profiling methods, including amplified ribosomal DNA restriction analysis, automated ribosomal intergenic spacer analysis, terminal restriction fragment length polymorphism, denaturing gradient gel electrophoresis, temperature gradient gel electrophoresis, single strand conformation polymorphism, and denaturing high-performance liquid chromatography. In this document, we present and critically compare these methods commonly used for the study of microbial diversity.

A novel bacterium was cultivated from an extreme thermal soil in Yellowstone National Park, Wyoming, USA, that at the time of sampling had a pH of 3.9 and a temperature range of 65-92 degrees C. This organism was found to be an obligate aerobic, non-spore-forming rod, and formed pink-colored colonies. Phylogenetic analysis of the 16S rRNA gene sequence placed this organism in a clade composed entirely of environmental clones most closely related to the phyla Chloroflexi and Thermomicrobia. This bacterium stained gram-positive, contained a novel fatty-acid profile, had cell wall muramic acid content similar to that of Bacillus subtilis (significantly greater than Escherichia coli), and failed to display a lipopolysaccharide profile in SDS-polyacrylamide gels that would be indicative of a gram-negative cell wall structure. Ultrastructure examinations with transmission electron microscopy showed a thick cell wall (approximately 34 nm wide) external to a cytoplasmic membrane. The organism was not motile under the culture conditions used, and electron microscopic examination showed no evidence of flagella. Genomic G+C content was 56.4 mol%, and growth was optimal at 67 degrees C and at a pH of 7.0. This organism was able to grow heterotrophically on various carbon compounds, would use only oxygen as an electron acceptor, and its growth was not affected by light. A new species of a novel genus is proposed, with YNP1(T) (T=type strain) being Thermobaculum terrenum gen. nov., sp. nov. (16S rDNA gene GenBank accession AF391972). This bacterium has been deposited in the American Type Culture Collection (ATCC BAA-798) and the University of Oregon Culture Collection of Microorganisms from Extreme Environments (CCMEE 7001).

Drinking water systems are known to harbour biofilms, even though these environments are oligotrophic and often contain a disinfectant. Control of these biofilms is important for aesthetic and regulatory reasons. Study of full-scale systems has pointed to several factors controlling biofilm growth, but cause-and-effect relationships can only be established in controlled reactors. Using laboratory and pilot distribution systems, along with a variety of bacterial detection techniques, insights have been gained on the structure and behaviour of biofilms in these environments. Chlorinated biofilms differ in structure from non-chlorinated biofilms, but often the number of cells is similar. The number and level of cellular activity is dependent on the predominant carbon source. There is an interaction between carbon sources, the biofilm and the type of pipe material, which complicates the ability to predict biofilm growth. Humic substances, which are known to sorb to surfaces, appear to be a usable carbon source for biofilms. The finding offers an explanation for many of the puzzling observations in full scale and laboratory studies on oligotrophic biofilm growth. Pathogens can persist in these environments as well. Detection requires methods that do not require culturing.