【正文】 er” solubilising agents than the polar detergents and they seem to have a much more limited ability to initiate the disruption of bacterial cells. Cell disruption by “physical” methods Bacteria whose cell walls are not susceptible to enzymatic and detergent treatments may be disrupted using “harsher” (., also on the DNA) methods which may be described, arbitrarily, as “physical” or “mechanical” [ 10,11,14,19]. Such methods generate DNA which is often sheared and usually not of the relatively uniform, large, molecular weight that can be attained using enzymatic and detergent disruption. Thus, such methods may not be appropriate for preparing DNA for specific analytical techniques. However, in instances wherein it has not been critical that the DNA be of uniform high molecular weight, methods employing a French pressure cell or a sonicator have been used with success. The use of glass particles with the (mini)bead beater is particularly effective for disrupting most bacteria and is the method of choice for the preparation of DNA from bacterial cells in problematic matrices (., soils) [23]. Additionally, a method for the production of high molecular weight DNA from Grampositive and acidfast bacteria using a microwave oven has been described [1]. However, the efficacies of such methods, all of which require additional, specialised, equipment, have been limited, in most cases, in the range of bacteria for which a given method can be applied. A further application which has been shown to be effective, particularly in bination with other steps, for disrupting extremely recalcitrant bacteria is the freeze (in liquid nitrogen) and fast thaw (at 95– 98176。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。Molecular Microbial Ecology Manual 1. Simplified protocols for the preparation of genomic DNA from bacterial cultures 2. Extraction of ribosomal RNA from microbial cultures 3. Extraction of microbial DNA from aquatic sources: Marine environments 4. Extraction of microbial DNA from aquatic sources: Freshwater 5. Methods for extracting DNA from microbial mats and cultivated microanisms: high molecular weight DNA from French press lysis 6. Extraction of microbial DNA from aquatic sediments 7. Extraction of microbial RNA from aquatic sources: Marine environments 8. Extraction of total RNA and DNA from bacterioplankton 9. Methods for extracting RNA or ribosomes from microbial mats and cultivated microanisms 10. Cell extraction method 11. DNA and RNA extraction from soil 12. Rapid simultaneous extraction of DNA and RNA from bulk and rhizosphere soil 13. Direct Extraction of Fungal DNA from Soil 14. Purification of microbial genes from soil and rhizosphere by magic capture hybridization and subsequent amplification of target genes by PCR 15. Direct ribosome isolation from soil 16. DNA Extraction from Actinorhizal Nodules 17. Quantification of nucleic acids 18. Quantification of nucleic acids from aquatic environments by using greenfluorescent dyes and microtiter plates 19. Degradation and turnover of extracellular DNA in marine sediments 20. Incorporation of thymidine into DNA of soil bacteria 21. Preparation of radioactive probes 22. Detection of Nucleic Acids by Chemiluminescence 23. Parameters of nucleic acid hybridization experiments 24. Detection and quantification of microbial DNA sequences in soil by Southern and dot/slot blot hybridization 25. Detection of microbial DNA sequences by colony hybridization 26. Polymerase chain reaction analysis of soil microbial DNA 27. Detection of microbial nucleic acids by polymerase chain reaction in aquatic samples 28. Isolation and detection of bacterial DNA sequences in dairy products 29. Quantitative PCR of environmental samples 30. Molecular beacons for homogeneous realtime monitoring of amplification products 31. Detection and enumeration of soil bacteria using the MPNPCR technique 32. Detection of mRNA and rRNA via reverse transcription and PCR in soil 33. Amplification of ribosomal RNA sequences 34. Cloning 16S rRNA genes and utilization to type bacterial munities 35. SARST, Serial Analysis of Ribosomal Sequence Tags 36. Oligonucleotide Fingerprinting of Ribosomal RNA Genes (OFRG) 37. Genotyping of bacterial isolates from the environment using LowMolecularWeight RNA fingerprints 38. Characterization of the diversity of ecologically important microbes by repPCR genomic fingerprinting 39. Genomic Fingerprinting o