Nucleic acid-based fluorescent probes in microbial ecology: application of flow cytometry

Microorganisms in natural environments have often been treated as 'black box' systems. Researchers have measured the inputs and outputs of the box, and have made bulk measurements on cell behaviour. However, unravelling the details of the diversity and interactions that exist within these microbial populations has proven exceptionally difficult. The information gained from the black box approach has been invaluable, and has allowed models of global foodwebs to be generated and tested. However, there is still little information about the interactions of individual microbial cells within natural populations. Such studies are essential to fully understand the integrated functioning of ecosystems. To achieve this goal, researchers need to be able to identify individual cells within a population, enumerate them, estimate both viability and activity, and monitor changes in response to relevant parameters. Due to the diversity, heterogeneity and numbers of cells that make up these populations, these measurements require automation and speed. At present, the use of flow cytometry in conjunction with nucleic acid probes provides an excellent method with which to pursue such studies.     

Rapid assessment of bacterial viability and vitality by rhodamine 123 and flow cytometry

A.S. KAPRELYANTS AND D.B. KELL. 1992. The fluorescent dye rhodamine 123 (Rh 123) is concentrated by microbial cells in an uncoupler-sensitive fashion. Steady-state fluorescence measurements with Micrococcus luteus indicated that provided the added dye concentration is below approximately 1 mmol/1, uptake is fully uncoupler-sensitive and is not accompanied by significant self-quenching of the fluorescence of accumulated dye molecules. 'Viable' and 'non-viable' cells are easily and quantitatively distinguished in a flow cytometer by the extent to which they accumulate the dye. The viability of a very slowly growing chemostat culture of Mic. luteus is apparently only about40–50%, as judged by plate counts, but most of the 'non-viable' cells can be resuscitated by incubation of the culture in nutrient medium before plating. The extent to which individual cells accumulate rhodamine 123 can be rapidly assessed by flow cytometry, and reflects the three distinguishable physiological states exhibited by the culture ('non-viable', 'viable' and 'non-viable-but-resuscitable'). Gram-negative bacteria do not accumulate rhodamine 123 significantly because their outer membrane is not permeable to it; a simple treatment overcomes this. Flow cytometry using rhodamine 123 should prove of general utility for the rapid assessment of microbial viability and vitality.     

Flow cytometric analysis of the cellular DNA content of Salmonella typhimurium and Alteromonas haloplanktis during starvation and recovery in seawater.

Flow cytometry was used to investigate the heterogeneity of the DNA content of Salmonella typhimurium and Alteromonas haloplanktis cells that were starved and allowed to recover in seawater. Hoechst 33342 (bisbenzimide) was used as a DNA-specific dye to discriminate between DNA subpopulations. The DNA contents of both strains were heterogeneous during starvation. S. typhimurium cells contained one or two genomes, and A. haloplanktis cells contained up to six genomes. S. typhimurium genomes were fully replicated at the onset of starvation. Each replication cycle was completed in the early stage of starvation for A. haloplanktis by stopping cells in the partition step of the cell cycle prior to division. Multigenomic marine cells can undergo rapid cell division without DNA synthesis upon recovery, resulting in large fluctuations in the DNA contents of individual cells. In contrast, the heterogeneity of the DNA distribution of S. typhimurium cells was preserved during recovery. The fluctuations in the DNA fluorescence of this strain seem to be due to topological changes in DNA. Flow cytometry may provide a new approach to understanding dynamic and physiological changes in bacteria by detecting cellular heterogeneity in response to different growth conditions.     

Feeding heterogeneity in ciliate populations: Effects of culture age and nutritional state

We have used a novel approach in conjunction with flow cytometry to quantify the biological heterogeneity of populations of the ciliate Tetrahymena pyriformis. It was found that the rate of particle uptake of exponentially growing cells is not uniform among cells and partially correlated with cell size. The physiological state and growth history of the culture was found to affect to a large degree the population's feeding heterogeneity. Stationary phase populations exhibited more uniform feeding behavior, as cell aging affects all cells and effectively reduces their ability to feed. Cells that were removed from the growth medium and resuspended in nonnutritive medium exhibited a more heterogeneous feeding behavior. The starved cells were stimulated to feed at considerably higher rates, and the stimulatory effect was more pronounced for larger cells. It is therefore demonstrated that population heterogeneity has to be evaluated in conjunction with the populations growth state as it is determined by the history of the population's growth and nutritional state. (c) 1994 John Wiley & Sons, Inc.     

TLM-Quant: An Open-Source Pipeline for Visualization and Quantification of Gene Expression Heterogeneity in Growing Microbial Cells

Gene expression heterogeneity is a key driver for microbial adaptation to fluctuating environmental conditions, cell differentiation and the evolution of species. This phenomenon has therefore enormous implications, not only for life in general, but also for biotechnological applications where unwanted subpopulations of non-producing cells can emerge in large-scale fermentations. Only time-lapse fluorescence microscopy allows real-time measurements of gene expression heterogeneity. A major limitation in the analysis of time-lapse microscopy data is the lack of fast, cost-effective, open, simple and adaptable protocols. Here we describe TLM-Quant, a semi-automatic pipeline for the analysis of time-lapse fluorescence microscopy data that enables the user to visualize and quantify gene expression heterogeneity. Importantly, our pipeline builds on the open-source packages ImageJ and R. To validate TLM-Quant, we selected three possible scenarios, namely homogeneous expression, highly ‘noisy’ heterogeneous expression, and bistable heterogeneous expression in the Gram-positive bacterium Bacillus subtilis. This bacterium is both a paradigm for systems-level studies on gene expression and a highly appreciated biotechnological ‘cell factory’. We conclude that the temporal resolution of such analyses with TLM-Quant is only limited by the numbers of recorded images.     

Viability and membrane potential analysis of Bacillus megaterium cells by impedance flow cytometry

Single cell analysis is an important tool to gain deeper insights into microbial physiology for the characterization and optimization of bioprocesses. In this study a novel single cell analysis technique was applied for estimating viability and membrane potential (MP) of Bacillus megaterium cells cultured in minimal medium. Its measurement principle is based on the analysis of the electrical cell properties and is called impedance flow cytometry (IFC). Comparatively, state-of-the-art fluorescence-based flow cytometry (FCM) was used to verify the results obtained by IFC. Viability and MP analyses were performed with cells at different well-defined growth stages, focusing mainly on exponential and stationary phase cells, as well as on dead cells. This was done by PI and DiOC(2)(3) staining assays in FCM and by impedance measurements at 0.5 and 10 MHz in IFC. In addition, transition growth stages of long-term cultures and agar plate colonies were characterized with both methods. FCM and IFC analyses of all experiments gave comparable results, quantitatively and qualitatively, indicating that IFC is an equivalent technique to FCM for the study of physiological cell states of bacteria.     

Current and future applications of flow cytometry in aquatic microbiology

Flow cytometry has become a valuable tool in aquatic and environmental microbiology that combines direct and rapid assays to determine numbers, cell size distribution and additional biochemical and physiological characteristics of individual cells, revealing the heterogeneity present in a population or community. Flow cytometry exhibits three unique technical properties of high potential to study the microbiology of aquatic systems: (i) its tremendous velocity to obtain and process data; (ii) the sorting capacity of some cytometers, which allows the transfer of specific populations or even single cells to a determined location, thus allowing further physical, chemical, biological or molecular analysis; and (iii) high-speed multiparametric data acquisition and multivariate data analysis. Flow cytometry is now commonly used in aquatic microbiology, although the application of cell sorting to microbial ecology and quantification of heterotrophic nanoflagellates and viruses is still under development. The recent development of laser scanning cytometry also provides a new way to further analyse sorted cells or cells recovered on filter membranes or slides. The main infrastructure limitations of flow cytometry are: cost, need for skilled and well-trained operators, and adequate refrigeration systems for high-powered lasers and cell sorters. The selection and obtaining of the optimal fluorochromes, control microorganisms and validations for a specific application may sometimes be difficult to accomplish.     

Using flow cytometry to quantify microbial heterogeneity

Flow cytometry is a powerful technique for the study of single cells, and thus it is of particular utility in the study of heterogeneity in microbial populations. This review seeks to highlight the role of flow cytometric analyses in studies of microbial heterogeneity, drawing wherever possible on recently published research articles. Whilst microbial heterogeneity is well documented in both natural and laboratory environments, the underlying causes are less well understood. Possible sources for the heterogeneity that is observed in microbial systems are discussed, together with the flow cytometric tools that aid its study. The role of flow cytometry in molecular biology is discussed with reference to gene reporter systems, which enable heterogeneity of gene expression to be monitored. With the recent sequencing of a variety of microbial genomes, it is anticipated that flow cytometry will have an increasing role to play in studying the effects of gene expression and mutation on heterogeneity, and in resolving the interactions of genetics and physiology.     

An industrial application of multiparameter flow cytometry: assessment of cell physiological state and its application to the study of microbial fermentations.

BACKGROUND: When using traditional microbiological techniques to monitor cell proliferation and viability, stressed, sublethally injured, or otherwise "viable but nonculturable" cells often go undetected. Because of this, such cells often are not considered by mathematical models used to predict bioprocess performance on scale-up and inaccuracies result. Therefore, analytical techniques, decoupled from postsampling growth, are desirable to rapidly monitor individual cell physiologic states during microbial fermentations. METHODS: Microbial cells, including Escherichia coli, Rhodococus sp., and Sacharomyces cerevisiae, were taken at various stages from a range of fermentation processes and stained with one of three mixtures of fluorescent stains: rhodamine 123/propidium iodide, bis-oxonol/propidium iodide, or bis-oxonol/ethidium bromide/propidium iodide. An individual cell's physiologic state was assessed with a Coulter Epics Elite analyzer based on the differential uptakes of these fluorescent stains. RESULTS: It was possible to resolve an individual cell's physiologic state beyond culturability based on the functionality of dye extrusion pumps and the presence or absence of an intact polarized cytoplasmic membrane, enabling assessment of population heterogeneity. This approach allows the simultaneous differentiation of at least four functional subpopulations in microbial populations. CONCLUSIONS: Fluorescent staining methods used in our laboratories have led to a functional classification of the physiological state of individual microbial cells based on reproductive activity, metabolic activity, and membrane integrity. We have used these techniques extensively for monitoring the stress responses of microorganisms in such diverse areas as bioremediation, biotransformation, food processing, and microbial fermentation; microbial fermentation is discussed in this article.     

Quantifying heterogeneity: flow cytometry of bacterial cultures

Flow cytometry is a technique which permits the characterisation of individual cells in populations, in terms of distributions in their properties such as DNA content, protein content, viability, enzyme activities and so on. We review the technique, and some of its recent applications to microbiological problems. It is concluded that cellular heterogeneity, in both batch and continuous axenic cultures, is far greater than is normally assumed. This has important implications for the quantitative analysis of microbial processes.     

Dead or Alive: Molecular Assessment of Microbial Viability

Nucleic acid-based analytical methods, ranging from species-targeted PCRs to metagenomics, have greatly expanded our understanding of microbiological diversity in natural samples. However, these methods provide only limited information on the activities and physiological states of microorganisms in samples. Even the most fundamental physiological state, viability, cannot be assessed cross-sectionally by standard DNA-targeted methods such as PCR. New PCR-based strategies, collectively called molecular viability analyses, have been developed that differentiate nucleic acids associated with viable cells from those associated with inactivated cells. In order to maximize the utility of these methods and to correctly interpret results, it is necessary to consider the physiological diversity of life and death in the microbial world. This article reviews molecular viability analysis in that context and discusses future opportunities for these strategies in genetic, metagenomic, and single-cell microbiology.     

The use of multi-parameter flow cytometry to compare the physiological response of Escherichia coli W3110 to glucose limitation during batch, fed-batch and continuous culture cultivations.

Multi-parameter flow cytometric techniques have been developed for the 'at-line' study of bacterial cultivations. Using a mixture of specific fluorescent stains it is possible to resolve an individual cells physiological state beyond culturability, based on the presence or absence of an intact polarised cytoplasmic membrane, enabling assessment of population heterogeneity. It has been shown that during the latter stages of small-scale (5 l), well mixed fed-batch cultivations there is a considerable drop in cell viability, about 17%, as characterised by cytoplasmic membrane depolarisation and permeability. These phenomena are thought to be due to the severe and steadily increasing stress associated with glucose limitation at high cell densities, during the fed-batch process. Such effects were not found in either batch or continuous culture cultivations. The possibility of using these findings for improved process control using 'on-line' flow cytometry are discussed.     

Characterization of intracellular accumulation of poly-beta-hydroxybutyrate (PHB) in individual cells of Alcaligenes eutrophus H16 by flow cytometry

Poly-beta-hydroxybutyrate (PHB) accumulates in individual cells of Alcaligenes eutrophus in the form of refractile bodies which alter the light-scattering properties of individual cells. Flow cytometry has been applied to measure the distributions of single-cell light-scattering intensity in Alc. eutrophus populations during batch cultivation of the organism. These measurements clearly identify heterogeneities in the inoculum which influence the lag interval prior to beginning of exponential growth. Light-scattering distributions show greater homogeneity and are extremely similar during balanced, exponential growth. After exhaustion of the nitrogen source and with carbon source still available, significant PHB accumulations occur and the flow cytometry measurements reveal extreme heterogeneity in single-cell light-scattering properties. These measurements clearly demonstrate the potential advantages of single-cell light-scattering measurements by flow cytometry for analysis and control of certain fermentation processes. Single-cell light-scat light-scattering measurements in conjunction with flow sorting instrumentation have been applied to demonstrate enrichment of PHB-producing cells, initially present in a number concentration of 0.01%by a factor of 300 in a single pass. Flow cytometry-cell sorting technology should find significant application in strain improvement and mutant selection.     

流式细胞术揭示出枯草芽孢杆菌多态异质性

新近的研究发现,微生物群体异质性现象普遍存在,与微生物群体许多关键功能密切相关.微生物群体中的多种异质性状态需要单细胞水平的分析技术才能被揭示,流式细胞术是获取异质性状态精确分布的重要工具.但微生物细胞尺寸微小、生物分子含量少、常常缺乏特异性试剂等都限制着传统流式细胞技术在微生物研究领域的应用.本论文采用新型的低背景、高灵敏度和高分辨率流式细胞仪,以增强的前向散射光、侧向散射光以及紫外光激发的细菌自发荧光水平这三个无需任何荧光标记就可以检测的信号为参数,首次揭示出不同生长状态的枯草芽孢杆菌具有复杂、动态的异质性状态分布.这一方法鉴定出的枯草芽孢杆菌多种状态及其与生理功能相关的、高度关联的变化,可能对该菌的生理变化规律及其分子机理的认识提供新的机遇.本论文也讨论了这一采用新型高灵敏度、高分辨率流式细胞仪测量非标记细胞参数的方法对于广泛开展各种微生物多态性研究具有巨大潜力.     

Heterogeneity in the rate of benzo[a]pyrene metabolism in single cells: quantitation using flow cytometry

We describe a method for quantitating heterogeneity in the rate of benzo[a]pyrene metabolism in single cells by using flow cytometry. We have used the technique to study the response of Hepa-1c1c7 mouse hepatoma cells to the microsomal enzyme inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin. Cells responded in a relatively homogeneous fashion at different times of induction with a maximally inducing concentration of the inducer. However, the induction response could be heterogeneous at a submaximal inducer concentration. We found even higher heterogeneity of enzyme activity among low-activity variants derived from the Hepa-1c1c7 cell line. When cells of either high or low activity were isolated from such a clonal population, propagated, and reanalyzed, they displayed average enzyme activity and heterogeneity identical to the parental cells; therefore, the heterogeneity represents transient, nonheritable differences between cells within the population.     

Development of a Robust Flow Cytometric Assay for Determining Numbers of Viable Bacteria

Several fluorescent probes were evaluated as indicators of bacterial viability by flow cytometry. The probes monitor a number of biological factors that are altered during loss of viability. The factors include alterations in membrane permeability, monitored by using fluorogenic substrates and fluorescent intercalating dyes such as propidium iodide, and changes in membrane potential, monitored by using fluorescent cationic and anionic potential-sensitive probes. Of the fluorescent reagents examined, the fluorescent anionic membrane potential probe bis-(1,3-dibutylbarbituric acid)trimethine oxonol [DiBAC(inf4)(3)] proved the best candidate for use as a general robust viability marker and is a promising choice for use in high-throughput assays. With this probe, live and dead cells within a population can be identified and counted 10 min after sampling. There was a close correlation between viable counts determined by flow cytometry and by standard CFU assays for samples of untreated cells. The results indicate that flow cytometry is a sensitive analytical technique that can rapidly monitor physiological changes of individual microorganisms as a result of external perturbations. The membrane potential probe DiBAC(inf4)(3) provided a robust flow cytometric indicator for bacterial cell viability.     

Separation, Identification, and Characterization of Microorganisms by Capillary Electrophoresis

The use of capillary electrophoresis (CE) for the analysis, identification, and characterization of microorganisms has been gaining in popularity. The advantages of CE, such as small sample requirements, minimal sample preparation, rapid and simultaneous analysis, ease of quantitation and identification, and viability assessment, make it an attractive technique for the analysis of microbial analytes. As this instrumental method has evolved, higher peak efficiencies have been achieved by optimizing CE conditions, such as pH, ionic strength, and polymer additive concentration. Experimental improvements have allowed better quantitation and more accurate results. Many practical applications of this technique have been investigated. Viability and identification of microbes can be accomplished in a single analysis. This is useful for evaluation of microbial analytes in consumer products. Diagnosis of microbe-based diseases is now possible, in some cases, without the need for culture methods. Microbe-molecule, virus-antibody, or bacteria-antibiotic interactions can be monitored using CE, allowing for the screening of possible drug candidates. Fermentation can be monitored using this system. This instrumental approach can be adapted to many different applications, including assessing the viability of sperm cells. Progress has been made in the development of microelectrophoresis instrumentation. These advances will eventually allow the development of small, dedicated devices for the rapid, repetitive analyses of specific microbial samples. Although these methods may never fully replace traditional approaches, they are proving to be a valuable addition to the collection of techniques used to analyze, quantitate, and characterize microbes. This review outlines the recent developments in this rapidly growing field.     

Separation, identification, and characterization of microorganisms by capillary electrophoresis.

The use of capillary electrophoresis (CE) for the analysis, identification, and characterization of microorganisms has been gaining in popularity. The advantages of CE, such as small sample requirements, minimal sample preparation, rapid and simultaneous analysis, ease of quantitation and identification, and viability assessment, make it an attractive technique for the analysis of microbial analytes. As this instrumental method has evolved, higher peak efficiencies have been achieved by optimizing CE conditions, such as pH, ionic strength, and polymer additive concentration. Experimental improvements have allowed better quantitation and more accurate results. Many practical applications of this technique have been investigated. Viability and identification of microbes can be accomplished in a single analysis. This is useful for evaluation of microbial analytes in consumer products. Diagnosis of microbe-based diseases is now possible, in some cases, without the need for culture methods. Microbe-molecule, virus-antibody, or bacteria-antibiotic interactions can be monitored using CE, allowing for the screening of possible drug candidates. Fermentation can be monitored using this system. This instrumental approach can be adapted to many different applications, including assessing the viability of sperm cells. Progress has been made in the development of microelectrophoresis instrumentation. These advances will eventually allow the development of small, dedicated devices for the rapid, repetitive analyses of specific microbial samples. Although these methods may never fully replace traditional approaches, they are proving to be a valuable addition to the collection of techniques used to analyze, quantitate, and characterize microbes. This review outlines the recent developments in this rapidly growing field.     

Assessment of cell concentration and viability of isolated hepatocytes using flow cytometry

The assessment of cell concentration and viability of freshly isolated hepatocyte preparations has been traditionally performed using manual counting with a Neubauer counting chamber and staining for trypan blue exclusion. Despite the simple and rapid nature of this assessment, concerns about the accuracy of these methods exist. Simple flow cytometry techniques which determine cell concentration and viability are available yet surprisingly have not been extensively used or validated with isolated hepatocyte preparations. We therefore investigated the use of flow cytometry using TRUCOUNT Tubes and propidium iodide staining to measure cell concentration and viability of isolated rat hepatocytes in suspension. Analysis using TRUCOUNT Tubes provided more accurate and reproducible measurement of cell concentration than manual cell counting. Hepatocyte viability, assessed using propidium iodide, correlated more closely than did trypan blue exclusion with all indicators of hepatocyte integrity and function measured (lactate dehydrogenase leakage, cytochrome p450 content, cellular ATP concentration, ammonia and lactate removal, urea and albumin synthesis). We conclude that flow cytometry techniques can be used to measure cell concentration and viability of isolated hepatocyte preparations. The techniques are simple, rapid, and more accurate than manual cell counting and trypan blue staining and the results are not affected by protein-containing media.