Detection and isolation of microorganisms in the environment

As we emphasised in the last chapter, microorganisms rarely, if ever, exist in nature as pure cultures but rather form mixed populations. Methods are required, therefore, for the detection and isolation of specific microbial types from such mixtures. The traditional method of isolation is the use of an enrichment culture, as described in Chapter 4. As examples, aerobic incubation with a supply of nitrite would assist in the isolation of nitrifying bacteria such as Nitrobacter from mud or sewage, whilst a minimal medium containing FeSO4 at pH 2 would encourage the isolation of A. ferrooxidans from a water sample.

We now know however that there are many types of microorganism in the environment that have so far resisted all attempts to culture them in the laboratory (often referred to as viable but non-culturable). The use of modern molecular techniques has helped us to identify the existence of a much broader range of bacteria and archaea than had previously been thought to exist. The extreme sensitivity of such methods means that we are able to demonstrate the presence of even a single copy of a particular bacterium in a mixed population. One such technique is called fluorescence in situ hybridisation (FISH). This uses a probe comprising a short sequence of single-stranded DNA or RNA that is unique to a particular microorganism, attached to a fluorescent dye. The microorganisms are fixed to a glass slide and incubated with the probe. The rules of base pairing in nucleic acids mean that the probe will seek out its complementary sequence, and cells carrying this sequence can be visualised under a fluorescence microscope. The most commonly used 'target' is ribosomal RNA, since this shows sequence variation from one microbial type to another, and because there are multiple copies within each cell, providing a stronger response. The polymerase chain reaction (PCR, Chapter 12) is another valuable tool in the identification of specific nucleic acid sequences. Other methods, not dependent on DNA, include the use of fluorescence-labelled antibodies raised against specific microorganisms.

Antibodies are proteins produced by the immune systems of higher animals in response to infection by a foreign organism; their main characteristic is their extreme specificity, thus they can be used to locate a specific protein.

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