This report is a review of the methods for the extraction, detection and identification of microorganisms in the environment. The report was produced by WS Atkins Environment as part of the research programme of the Chemicals and Biotechnology Division of the Department of Environment, Food and Rural Affairs (DEFRA), and is a revised and updated version of a report originally prepared by the University of Liverpool.

The reason for the study is that genetically modified microorganisms may be released and we want to know how to monitor and detect them. The original methods for the detection and identification of microorganisms were culture-based techniques, where the microorganism was grown (cultured) on a medium containing substrates specific to its own nutritional requirements. Such techniques required the microorganism to be extracted from its environment before any identification could be made. The requirement for extraction is used as the principle differentiating factor between detection/identification techniques. Methods that require an extraction stage are described as ex-situ, and those that do not require the microorganism to be extracted from its environment are known as in-situ. However, in-situ methods may involve some extraction or sample clean-up step prior to the detection or identification method. In-situ methods are divided into microscope, colorimetric and mo lecular-based approaches.

Because of the diversity of microorganisms in the environment, and the heterogeneity of many environments at a microbial level, a large number of different methods have been developed to detect and/or identify as large a proportion of the microorganisms present as possible. However, due to the diversity of microorganisms, and the different niches in which they inhabit, no single method is applicable for the extraction, detection or identification of all the microorganisms in a particular sample (except for clinical samples which may be infected with a single species of microorganism). The detection and/or identification of the microorganisms present in many environmental samples is also restricted by the existence of many of the microorganisms in the environment in a viable but non-culturable state (VBNC). VBNC microorganisms cannot be isolated by culture-based methods, and require the ap plication of in-situ methods.

• fungi: assessment, sampling and analysis