Analysis of bacterial function by multi-colour fluorescence flow cytometry and single cell sorting

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Abstract

With the increased awareness of the problems associated with the growth dependent analysis of bacterial populations, direct optical detection methods such as flow cytometry have enjoyed increased popularity over the last few years. Among the analyses discussed here are: (1) Bacterial discrimination from other particles on the basis of nucleic acid staining, using sample disaggregation to provide fast reliable enumeration while minimizing data artefacts due to post sampling growth; (2) Determination of basic cell functions such as reproductive ability, metabolic activity and membrane integrity, to characterise the physiological state or degree of viability of bacteria; and (3) The use of single cell sorting onto agar plates, microscope slides or into multi-well plates to correlate viability as determined by cell growth with fluorescent labelling techniques. Simultaneous staining with different fluorochromes provides an extremely powerful way to demonstrate culture heterogeneity, and also to understand the functional differences revealed by each stain in practical applications. Analysis of bacterial fermentations showed a considerable drop (20%) in membrane potential and integrity during the latter stages of small scale (5L), well mixed fed-batch fermentations. These changes, not found in either batch or continuous culture fermentations, are probably due to the severe, steadily increasing stress associated with glucose limitation during the fed-batch process, suggesting ‘on-line’ flow cytometry could improve process control. Heat injured cells can already show up to 4 log of differences in recovery in different pre-enrichment media, thus contributing to the problem of viable but non-culturable cells (VBNC’s). Cytometric cell sorting demonstrated decreasing recovery with increasing loss of membrane function. However, a new medium protecting the cells from intracellular and extracellular causes of oxidative stress improved recovery considerably. Actively respiring cells showed much higher recovery improvement than the other populations, demonstrating for the first time the contribution of oxidative respiration to intracellular causes of damage as a key part of the VBNC problem. Finally, absolute and relative frequencies of one species in a complex population were determined using immunofluorescent labelling in combination with the analysis of cell function. The detail and precision of multiparameter flow cytometric measurements of cell function at the single cell level now raise questions regarding the validity of classical, growth dependent viability assessment methods.

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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