【正文】 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 crosslinking interbridges of the peptidoglycan layers of the cell walls of bacteria. The applicability of Proteinase K for disrupting bacterial cell walls is enhanced by its insensitivity to specific chelating agents, allowing it to be utilised in bination with EDTA and lysozyme. However, the peptide interbridges of the cell walls of different species, formed by different binations of ponent amino acids, with inherently different susceptibilities to cleavage, may be more or less resistant to Proteinase K lysis. While lysozyme and proteinase K are, probably, the enzymes most monly used for the disruption of bacterial cells, additional bacterial celldisrupting enzymes also have been reported with broad or narrow specificities. Other muramidases, mutanolysin and lysostaphin react, analogous to lysozyme, at the peptide linkages in the cell walls, although the species which are susceptible to these enzymes differ from those which are affected by lysozyme [2, 20, 26]. Subtilisins are extracellular proteases, produced by Bacillus spp., exhibiting a broad specificity in hydrolysing most peptide and ester bonds [24]. They are not inactivated by chelating agents, which makes them applicable in bination with EDTA. The application of achromopeptidase has been limited to the disruption of Grampositive cells, principally staphylococci [9], although applications with other bacteria have been reported. Cell disruption by detergent treatments Detergents provide effective, yet relatively gentle, means for disrupting cells, binding strongly to proteins and causing irreversible denaturation. Further, conditions which cause dissociation of protein (., high pH, low and high ionic strength, etc.) tend to enhance, as well, the solubilisation efficiencies of detergents [7]. Detergents are particularly effective for disrupting bacteria when their cell walls have been damaged (., through the actions of metal chelating agents, lysozyme and Proteinase K) prior to their addition to the cell suspension. Sodium dodecyl sulfate (SDS) is an anionic detergent which reacts, at low concentrations, at protein hydrophobic sites, binding cellular proteins and lipoproteins, forming SDSpolypeptide micellar plexes, and effectively denaturing them and promoting the dissociation of nucleic acids [17]. Further, SDS inhibits nucleases and does not interact with the hydrophilic nucleic acids. Some proteins form SDS plexes only after they have been heated or treated with reagents (., mercaptoethanol) to cleave intraprotein disulfide bonds. Nlauroylsarcosine (Sarcosyl), empirically, may be more effective at denaturing cellular polysaccharide material and can be used, instead of SDS, for the disruption of bacterial cells (., Azotobacter, Beijerinckia, Klebsiella, etc.) which produce copious amounts of capsule. Cetyltrimethyl ammonium bromide (CTAB), a cationic detergent, has been used extensively in the preparation of nucleic acids from fungi and plants, when large amounts of polysaccharide materials tend to interfere with the extraction. However, CTAB also has been proven useful for DNA extractions from bacterial cells by denaturing and precipitating the cell wall lipopolysaccharides and proteins [12]. In the presence of monovalent cation (., Na+) concentrations above M, DNA will remain soluble. Nonpolar detergents, including the Triton X series, Tween series, Nonidet P40, etc., are generally “mild