Analysis of bacterial function by multi-colour fluorescence flow cytometry and single cell sorting
Section snippets
Microbial cytometry: bulk vs. single cell measurements
Because of the importance of microbiology to human health, methods have been developed to enumerate bacteria, to identify them and to look at the impact of physical, chemical or biological interventions. Bulk measurements, based on detection of changes in turbidity, conductivity, or gas pressure of liquid media (Fig. 1) have become popular for bacterial detection because of their speed and relative simplicity. Selective growth media can allow some degree of bacterial differentiation, but
Detection and counting of microorganisms
All microbial detection systems that rely on cell replication are limited by the requirement to grow bacteria in an artificial environment. Lack of symbiotic partners or an unsuitable micro-environment may result in inaccurate plate counts from natural samples. Also, some cells will only grow anaerobically, some only aerobically, and some under both conditions. Therefore representative counts, which ideally detect healthy, injured, dormant, and ‘viable but sometimes-non-culturable’, as well as
Classification of cell functionality.
Viability is the key cell function investigated in microbiology. Multi-colour flow cytometric analysis allows differentiation of stages far beyond the classical definition of viability, which is usually defined by demonstrable reproductive growth (Fig. 3). The fact that complex cellular functions, other than growth, can be detected, has lead to a shift in the interpretation of the term “viability”, thus generated the term of “viable but non-culturable cells”. The use of the functional criteria
Practical applications of the multi-colour assessment of cell functionality.
The staining combinations described above have been used to investigate biotechnological processes and deliberate cell injury. This section gives a brief summary of those applications.
Combined identification, counting and viability assessment
Identification of bacteria is classically based on morphology, nutritional requirements and metabolic reactions, which are only of limited use in direct single cell analysis. Serological methods based on antigenic differences have been used for a long time, and, more recently, genetic methods have been introduced into the field of bacterial identification. The advantages of methods using rRNA probes are the capability for broader classification and the ability to design probes from databases
Summary
Direct analysis of bacteria on a single cell level will continue to enhance our understanding of microbial populations, their heterogeneity and complexity. Flow cytometry in combination with single cell sorting has allowed the interpretation and verification of the properties of a number of fluorescent stains. In biotechnological applications, the multicolour staining approach provides important physiological information at the individual cell level about process efficiency that is almost
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