Introduction. Microbial Flow Cytometry: Technology and Applications

Martin G. Wilkinson

An introduction to flow cytometry. free download

1. Microbial Cytometry: What It Was, Is, and May Be

Howard M. Shapiro

Microbes were discovered, counted, and sized in a microfluidic device by Antonie van Leeuwenhoek in the late 1600s, but their importance in nature and in health did not become apparent until the late 1800s, when many classical methods of microbial analysis were developed. It has only been since the mid-1900s that cytometry has made it possible to detect, identify, and characterize single microorganisms and other cells in terms of their structure, function, and genetics. Modern multiparameter cytometry typically makes multiple physical measurements of a cell, most commonly of light scattering and fluorescence, which may be emitted from cellular constituents themselves or from reagents or probes added to facilitate their detection and quantification. Flow cytometers, in which cells are analyzed as they flow through the apparatus in a fluid stream, can be equipped with sorting capability to allow separation of cells with preselected characteristics; however, when sorting is not required, an increasing proportion of the measurements now made in flow cytometers can be made in simpler, less expensive imaging systems. By 2020, improvements along this line should allow the benefits of cytometric technology to be applied to problems and in places for and in which it was previously unaffordable. Microbiologists, who have been among the "have-nots" with respect to cytometry, even in affluent countries, stand to benefit substantially from this.

2. Non-Destructive On-Chip Imaging Cytometry Assay for Constructive On-Chip Cellomics Studies

Kenji Yasuda

We have developed two series of the novel non-destructive imaging cell cytometry assays exploiting microfabrication technology. The on-chip imaging flow cytometry system uses a 1/10,000 s ultra high speed camera with a real-time image analysis unit and a poly(methyl methacrylate (PMMA) based disposable microfluidic chip with the following features: (1) a fully automated system for application and collection of samples using 3-D micropipetting; (2) arrangement of the entire fluidic system in a disposable plastic chip for lining-up of biological samples in laminar flow using hydrodynamic focusing; (3) an optical system for direct observation-based cell identification with specific image indexes using phase-contrast/fluorescence microscopy and real-time image processing; (4) a non-destructive wider-size dynamic range sorting procedure using mild electrostatic force in a laminar flow using agarose gel electrodes to prevent loss of electrode or electrolysis bubble formation; (5) a contamination-free re-cultivation micro-reservoir for collected target cells, and (6) removable DNA aptamer labelling. On-chip single cell imaging cytometry has the advantage of being able to measure the ‘algebraic' inheritance of epigenetic character of single cells using imaging indexes. Exploiting these advantages, target cells can be successfully separated and evaluated and their characteristics by imaging. Results have shown that the on-chip imaging flow cytometry system and the on-chip single-cell imaging cytometry system are applicable for biological research and clinical diagnostics.

3. Application of Flow Cytometry to Environmental Biotechnology

Mónica Herrero and Mario Díaz

The number of publications applying Flow Cytometry (FCM) as an analytical technique is increasing in the field of environmental biotechnology. This field is defined "as the development, use and regulation of biological systems for remediation of contaminated environments (land, air, water) for environmental-friendly processes". FCM complements existing technologies and enables new information to be obtained from microbial ecosystems. Difficulties with analysis and differentiation of bacterial cells due to their small size and similar morphologies can be addressed with recent technical advances in equipment handling, improved instrument cell-sorting capacities, development of fluorescent dyes and multi-parametric analysis. FCM allows the use of taxonomic and physiological probes to detect and characterize microbial communities or single cells, even target cells expressing specific gene functions. Indeed many of the microorganisms present in natural samples or involved in environmental bioprocesses are not cultivable and often undergo changes in their conditions that rapidly affect their population dynamics. Characterization of microbial physiological states is a key application for environmental bioprocesses optimization. FCM has been used to ensure effectiveness of drinking water quality, wastewater treatments, to check the hazardous effects of anthropogenic toxic substances released to the environment or to assess bacterial viability in soils, facilitating rapid statistically relevant data acquisition, within almost real-time conditions.

4. Flow Cytometry and Microbial Community Fingerprinting

Susann Müller and Kathleen Schleinitz

Flow cytometry (FCM) can be used to reveal community patterns by analyzing certain cell parameters that reflect microbial community structures. The method is valuable for separating a microbial community into sub-sets of individuals with shared physiological properties, such as proliferation activity and scatter characteristics. Fingerprinting tools elucidating community composition, i.e. describing the phylogenetic affiliation of the single community members can also complement FCM. These techniques are introduced with a short overview. Their applications for microbial community composition and structure analyses will be compared. The chapter then describes how fingerprinting techniques can be applied to characterize the FCM-separated subsets. This chapter does not provide detailed protocols for applying FCM to microbial communities, as these protocols will vary depending on the community and physiological parameters of interest. Instead, this chapter highlights the general methodology and approach and discusses potential future advances in the field.

5. Application of Flow Cytometry to the Detection of Pathogenic Bacteria

Deirdre Kennedy and Martin G. Wilkinson

Outbreaks of infections have emphasised the necessity for rapid and economic detection methods for pathogens in samples ranging from those of clinical origin to food products during production and retail storage and increasingly in environmental samples. Flow Cytometry (FCM) allows the rapid acquisition of multi-parametric data regarding cell populations within fluidised samples. However, the application of FCM to pathogen detection depends on the availability of specific fluorescent probes such as antibodies and RNA probes capable of detecting and isolating pathogens from these diverse samples. A particular issue for FCM methodology is the ability to recover and discriminate bacteria from the sample matrix which may pose a major technical hurdle towards accurate and sensitive analysis. This chapter focuses on detection of pathogens using FCM in samples originating from food, water, environmental and clinical sources and outlines the current state of the art and potential future applications.

6. The Use of Flow Cytometry to Study Sporeforming Bacteria

Ultan Cronin

Sporeformers comprise a diverse group of important bacteria and possess two unique traits: the ability to form dormant and resistant spores and their ability to restart metabolism and germinate. Flow cytometry (FCM) offers many possibilities for the study of sporeformers, bringing advantages in terms of speed, novelty and the ability to measure heterogeneity within a population. In this chapter, five categories of FCM studies involving sporeformers (mainly involving species within the genera, Bacillus and Clostridium) have been identified: those based on autofluorescence or light scatter; those involving antibodies or fluorescent in situ hybridization probes; those using reporter proteins in the context of genetic transformation; those involving permeability dyes; those utilizing physiological dyes. In the past, instrumentation was expensive, difficult to operate and the range of fluorescent dyes and antibodies available to the microbiologist was limited. In the last half a decade, however, the number of low-cost, easy-to-run and powerful (≥ two lasers, ≥ six detectors) digital instruments has mushroomed. In a similar vein, the variety of monoclonal antibodies and dyes available to the microbiologist is rapidly increasing. Both developments allow the researcher interested in sporeformers to design more information-rich and daring experiments than ever before. Experiments combining the use of fluorescent reporter proteins, physiological dyes, a permeability marker and a monoclonal antibody are now possible. Using antibodies or fluorescent dyes specific to particular structural components of endospores such as the exosporium or protein coat, FCM may become a very useful tool in the real-time analysis of sporulation or resistance mechanisms. FISH probes could be used for environmental studies. The growing variety of reporter proteins such as GFP or mCherry allows the possibility of simultaneously studying the expression of a number of genes, an experimental approach which may help unravel the sequence of events leading to sporulation. The use of multiplex bead arrays could prove a powerful tool in characterising a species' repertoire of secreted proteins or toxins. Of prime importance in implementing new methods for the study of sporeformers is their comparison with existing gold standard techniques. Even a research tool as powerful as FCM must be shown to yield data as robust and reliable as conventional methods.

7. Flow Cytometry of Yeasts and Other Fungi

Tim W. Overton

Flow cytometry is used extensively to investigate many aspects of yeast physiology and cell biology. This chapter describes the most common applications of flow cytometry and fluorescence-activated cell sorting (FACS) to yeasts and other fungi, and reviews recent studies to capture current directions in the field. Pure and applied aspects are discussed, with a focus on the areas of fundamental cell biology, medical applications, monitoring during bioprocesses including brewing, and the use of yeast as a molecular biology tool. Novel flow cytometric and microfluidic systems which utilise yeast as a model organism are also described and their relevance to previous research in the area and their possible application for future fundamental and applied research are discussed.

8. The Application of Flow Cytometry to the Study of Lactic Acid Bacteria Fermentations

I.A. Doolan, M. G. Wilkinson and D.K. Hickey

This chapter will focus on Lactic Acid Bacteria (LAB) and their importance in the production of a wide range of fermented foods as both viable and non-viable organisms. A comparison of traditional microbiological methods used to study LAB will be outlined as well as examples of how flow cytometry (FCM) can be successfully employed to analyse LAB in viable and non-viable states during a fermentation process such as cheese manufacture. This chapter also deals with some of the difficulties encountered with FCM analysis of certain food samples and how these issues may be overcome. The potential applications of FCM to the study of LAB in both academic and applied research issues are also discussed.

9. Flow Cytometry for Rapid Microbiological Analysis of Drinking Water: From Science to Practice, an Unfinished Story

Thomas Egli and Stefan Kötzsch

For many years routine microbiological analysis of drinking water has been dominated by plating methods. Established by Koch more than a century ago, the world-wide standards include both indicator organisms for faecal contamination as hygiene parameters (Escherichia coli, coliforms, enterococci), and the "heterotrophic plate count" (HPC) as a general parameter of microbiological water quality. These methods are slow, requiring 1-10 days before data become available. In particular, the HPC has been criticized over the past 30 years as underestimating the real number of microbial cells by two or three orders of magnitude. Flow cytometry offers a range of options to detect cells quickly, and we have recently explored the potential of a number of FCM-based methods for rapid (< 1h) microbiological analysis of drinking and surface waters. These include total cell counting (TCC), the ratio of small/big cells (LNA:HNA-ratio), community fingerprints, assessing cell viability and physiological state (all in 15min), or detection and quantification of selected pathogens (in < 2h). Determination of TCC and the LNA:HNA-ratio are now officially accepted and recommended parameters for freshwater analysis in Switzerland. Determination of the TCC by flow cytometry is already routinely used by a number of Swiss and Dutch drinking-water suppliers. FCM has been proven to allow fast and realistic assessment of water treatment processes and rapid quantification of microbial regrowth and bio(in)stability in drinking-water distribution networks and in public and private buildings. In addition, online monitoring of TCC has already been reported and will be commercially available shortly. The potential for applications of FCM-based methods in the field of drinking-water analysis is still far from being fully exploited.