【正文】 ing efficiency and interpretation of diversity in 16S rRNA gene libraries 77. LIBSHUFF Comparisons of 16S rRNA Gene Clone Libraries 78. Cluster analysis and statistical parison of molecular munity profile data 79. Computerassisted analysis of molecular fingerprint profiles and database construction 80. Multivariate statistical methods and artificial neural works for analysis of microbial munity molecular fingerprints 81. Quantitative fluorescence in situ hybridisation (FISH): statistical methods for valid cell counting 82. Oligonucleotide probe design for mixed microbial munity microarrays and other applications and important considerations for data analysis 83. Design of microarrays for genomewide expression profiling 84. Assessment of the membrane potential, intracellular pH and respiration of bacteria employing fluorescence techniques 85. Use of microelectrodes to measure in situ microbial activities in biofilms, sediments, and microbial mats 86. Application of wholecell biosensors in soil 87. Detection of bacterial homoserine lactone quorum sensing signals 88. BrdU Substrate Utilization Assay 89. Stable isotope probing of nucleic acids to identify active microbial populations 90. Linking microbial munity structure and functioning: stable isotope (13C) labeling in bination with PLFA analysis 91. Correlating singlecell count with function in mixed natural microbial munities through STARFISH 92. Differential display of mRNA 93. Macroarrays protocols for gene expression studies in bacteria 94. Oligonucleotidebased functional gene arrays for analysis of microbial munities in the environment 95. Proteomic Analysis of Bacterial Systems Molecular Microbial Ecology Manual Kluwer Academic Publishers2020 Section 1 Isolation of Nucleic Acids Simplified protocols for the preparation of genomic DNA from bacterial cultures Edward Moore1, Angelika Arnscheidt1, Ante Kr220。ger1, Carsten Str214。mpl1 and Margit Mau1 (1) Division of Microbiology, GBF – German National Research Centre for Biotechnology, Mascheroder Weg 1, D38124, Braunschweig, Germany Introduction The development of methodologies for the analysis of microanisms and microbial ecology, at the molecular level (., nucleic acids, proteins, lipids, and their genes), has progressed phenomenally in recent years. Each methodology has specific advantages and disadvantages, or plications. However, the advances in PCR, cloning, gene probing, sequencing and fingerprinting have enabled techniques exploiting nucleic acids to be utilised extensively for the analysis of microanisms. Often, such protocols require, firstly, that the nucleic acids are extracted in a form which can be employed for the analyses. This may, in some cases, be more difficult than anticipated initially, since many bacteria are extremely resistant to cell disruption. Typically, these are Grampositive bacteria (., Mycobacterium spp., Peptococcus spp., Rhodococcus spp., etc.), as well as some Archae (., methanogens), with thick cell walls of polysaccharide or pseudopeptidoglycan, and many species of fungi and algae. General considerations Several protocols have been developed and described for the preparation of genomic DNA from bacteria, beginning with the prototypal method of Marrnur [16], which involved: a) cell disruption by an enzymedetergent lysis。 b) extractions with anic solvents。 and c) recovery of the DNA by alcohol precipitation. Subsequent protocols have usually involved some modification of one or more of these general steps. Cell disruption The most difficult and uncertain step in obtaining DNA from bacterial cultures is that of disrupting the cells. The difficulties derive, in part, from imposed limitations in the handling of the preparations, which are necessary for obtaining genomic DNA of high molecular weight. Thus, in general, the most desirable means of disrupting bacterial cells for obtaining genomic DNA is throughenzymatic digestion and detergent lysis. Such a strategy is enhanced by prior treatment of cells with a metal chelating agent, such as ethylenediaminetetraacetic acid (EDTA). If the cell wall of the anism is susceptible to such treatments, relatively high molecularweight genomic DNA can be obtained which is applicable for a number of analytical techniques. Further, the lysis should be carried out in a buffered (pH 8– 9) medium containing EDTA. The alkaline pH reduces electrostatic interactions between DNA and basic proteins, assists in denaturing other cellular proteins and inhibits nuclease activities. EDTA binds divalent cations, particularly Mg2+ and Mn2+, reducing the stabilities of the walls and membranes and also inhibits nucleases which have a requirement for metal cations. Cell disruption by enzymatic treatments Lysozyme, isolated mercially from chicken egg white, is a member of the broad class of muramidases which catalyse the hydrolysis of the β 1,4glycosidic linkage between the Nacetylmuramic acidNacetylglucosamine repeating unit, prising a major part of the peptidoglycan layer of the cell walls of most bacteria [18]. Lysozyme is especially effective in disrupting bacterial cells when used in bination with EDTA [15]. Lysozyme and related enyzmes are useful for disrupting the cells of a broad range of bacterial species, although many species are not particularly susceptible to muramidase treatment due, presumably, to layers of protein or capsular slime, which protect the peptidoglycan. Additionally, as their cell walls do not contain peptidoglycan, all described species of Archae are resistant to lysozyme activity. Proteinase K, a serine protease produced by the fungus Tritirachium album, cleaves adjacent to the carboxyl groups of aliphatic and aromatic amino acids involved in peptide bonding [4], including those prising the peptide