Fluorescence staining and flow cytometry for monitoring microbial cells

Large numbers of microbiological samples are analysed annually using tradit ional culture-based techniques. These techniques take hours to days to yiel d a result, are tedious and are not suitable for non-culturable microorgani sms. Further, culture-based techniques do not provide real-time information on the physiological status of the organism in situ which is important in the industrial manufacture of many microbial products. Flow cytometry offer s the prospect of real-time microbial analysis of individual microorganisms , without dependency on microbial culture. However, flow cytometry has not been extensively used as a tool for routine microbial analysis. This has be en mainly due to thr:high cost and complexity of instrumentation, the need for trained flow cytometrists and the lack of assay kits with appropriate b iological reagents for specific applications. Many modern instruments are n ow relatively simple to operate, due to improvements in the user-interface, and no longer need a specialist operator. However. most cytometers are sti ll reliant on analogue technology first developed 20-30 years ago. The inco rporation of modern, solid state opto-electronics combined with micro-fabri cation and digital signal processing technology offers the prospect of simp le to use, low cost and robust instruments suitable for microbial analyses. Advances are being made in the development of a range of biological reagen ts and these are now being formulated into simple to use kits for microbiol ogical applications. Currently, these kits are largely restricted to simple analyses, for example to assay for total or viable numbers of microorganis ms present. However, technologies are available to selectively label specif ic types of microorganisms. For example, fluorescent antibodies can be used to label microorganisms according to expression of particular antigens, fl uorescent in situ hybridisation to label according to phylogeny and fluorog enic enzymatic substrates to label according to expression of specific enzy me activities. Reagents are also available that stain viruses sufficiently brightly to enable their direct detection in environments such as sea water . Microorganisms need to be detected in a variety of different matrices (e. g., water, mud, food, and beverages) and these matrices may be highly varia ble in nature (e.g., tap water compared to river water). Many matrices have high background autofluorescence (e.g., algae and minerals in water sample s) or may bind non-specifically to the fluorescent biological reagents used (e.g., protein micelles in milk). Formulation of biological reagents and s ample pre-treatments rue critical to the development of suitable microbiolo gical assays. Here, developments in instrumentation and biological reagents for microbiological applications are reviewed with specific examples from environmental or industrial microbiology. The broader considerations for th e development of microbial assays for how cytometry are also considered. (C ) 2000 Elsevier Science B.V. All rights reserved.