Evaluation of Ultrasound-Induced Damage to Escherichia coli and Staphylococcus aureus by Flow Cytometry and Transmission Electron Microscopy

As a nonthermal sterilization technique, ultrasound has attracted great interest in the field of food preservation. In this study, flow cytometry and transmission electron microscopy were employed to investigate ultrasound-induced damage to Escherichia coli and Staphylococcus aureus. For flow cytometry studies, single staining with propidium iodide (PI) or carboxyfluorescein diacetate (cFDA) revealed that ultrasound treatment caused cell death by compromising membrane integrity, inactivating intracellular esterases, and inhibiting metabolic performance. The results showed that ultrasound damage was independent of initial bacterial concentrations, while the mechanism of cellular damage differed according to the bacterial species. For the Gram-negative bacterium E. coli, ultrasound worked first on the outer membrane rather than the cytoplasmic membrane. Based on the double-staining results, we inferred that ultrasound treatment might be an all-or-nothing process: cells ruptured and disintegrated by ultrasound cannot be revived, which can be considered an advantage of ultrasound over other nonthermal techniques. Transmission electron microscopy studies revealed that the mechanism of ultrasound-induced damage was multitarget inactivation, involving the cell wall, cytoplasmic membrane, and inner structure. Understanding of the irreversible antibacterial action of ultrasound has great significance for its further utilization in the food industry.     

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.     

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.     

Population dynamics of a continuous fermentation of recombinant Saccharomyces cerevisiae using flow cytometry

The plasmid instability of genetically modified microorganisms during prolonged bioreactor operations is one of the major problems to be overcome in the production of recombinant proteins. The use of flow cytometry to monitor a fermentation process with recombinant cells in a CSTR is reported here. This technique has been applied to determine the fraction of plasmid-bearing cells (P+) of a recombinant Saccharomyces cerevisiae strain harboring the EXG1 gene in a continuous stirred tank bioreactor with a working volume of 2 L. The different levels in the expression of the EXG1 gene, which encodes the enzyme exo-beta-glucanase, were used to determine the P+ fraction. Other parameters such as viability, cellular protein, cell size and structure were also monitored using flow cytometry. This technique has two main advantages over the conventional method of determining the P+ fraction (plating in selective and non-selective solid media): (a) it takes a very short period of time to obtain a measurement that provides multiple parametric information; and (b) it is more representative of the bioreactor cell population since it can analyze thousands of cells in the same sample. A continuous operation (432 h) with the recombinant strain in a CSTR was carried out to test the application of this technique. Measurements of cellular exo-beta-glucanase activity and cellular protein content closely correlates to the measured fraction of plasmid-containing cells in the population. Moreover, the standard deviation of the fraction of P+ cells determined using this technique was very low (about 2%). Recombinant protein production also increased the size of the yeast cells, whereas the recombinant cells also had a more complex internal structure than the non-recombinant host strain.     

Fundamentals of flow cytometry

Flow cytomelers arc instruments that arc used primarily to measure the physical and biochemical characteristics of biological particles. This technology is used to perform measurements on whole cells as well as prepared cellular constituents, such as nuclei and organelles. Flow cytomcters are investigative tools for a broad range of scientific disciplines because they make measurements on thousands of individual cells/ particles in a matter of seconds. This is a unique advantage relative to other detection instruments that provide bulk particle measurements. Flow cytomety is a complex and highly technical field; therefore, a basic understanding of the technology is essential for all users. The purpose of this article is to provide fundamental information about the instrumentation used for flow cytometry as well as the methods used to analyze and interpret data. This information will provide a foundation to use flow cytometry effectively as a research tool.     

Flow cytometry and FACS applied to filamentous fungi

Flow cytometry is an automated, laser- or impedance-based, high throughput method that allows very rapid analysis of multiple chemical and physical characteristics of single cells within a cell population. It is an extremely powerful technology that has been used for over four decades with filamentous fungi. Although single cells within a cell population are normally analysed rapidly on a cell-by-cell basis using the technique, flow cytometry can also be used to analyse cell (e.g. spore) aggregates or entire microcolonies. Living or fixed cells can be stained with a wide range of fluorescent reporters to label different cell components or measure different physiological processes. Flow cytometry is also suited for measurements of cell size, interaction, aggregation or shape using non-labelled cells by means of analysing their light scattering characteristics. Fluorescence-activated cell sorting (FACS) is a specialized form of flow cytometry that provides a method for sorting a heterogeneous mixture of cells into two or more containers based upon the fluorescence and/or light scattering properties of each cell. The major advantage of analysing cells by flow cytometry over microscopy is the speed of analysis: thousands of cells can be analysed per second or sorted in minutes. Drawbacks of flow cytometry are that specific cells cannot be followed in time and normally spatial information relating to individual cells is lacking. A big advantage over microscopy is when using FACS, cells with desired characteristics can be sorted for downstream experimentation (e.g. for growth, infection, enzyme production, gene expression assays or ‘omics’ approaches). In this review, we explain the basic concepts of flow cytometry and FACS, define its advantages and disadvantages in comparison with microscopy, and describe the wide range of applications in which these powerful technologies have been used with filamentous fungi.     

Cytometric monitoring of growth, sporogenesis and spore cell sorting in Paenibacillus polymyxa (formerly Bacillus polymyxa)

AIMS: Formation of bacterial endospores is a basic process in Gram-positive bacteria and has implications for health, industry and the environment. Flow cytometry offers a practical alternative for the rapid detection, enumeration and characterization of bacterial endospores. METHODS AND RESULTS: Paenibacillus polymyxa was chosen for this study because its spores cause sporangium deformation and have thick walls with a star-shaped section. Sporulating populations were analysed with a particle analyser and a flow cytometer after labelling with propidium iodide and Syto-13. Flow cytometric detection of single spores was confirmed by optical and scanning electron microscopy after cell sorting. Four cell sub-populations were cytometrically detected in P. polymyxa cultures grown in liquid sporulation medium. Two sub-populations consisted of vegetative cells differing in both morphology and viability; the other two sub-populations consisted of spores differing in their viability. CONCLUSIONS: This work has shown that flow cytometry is a simple and fast method (less than 15 minutes for sample preparation and analysis) for the study of the sporulation in P. polymyxa. The use of this technique allowed both detection and quantification of sporulation inside a culture, and distinguished cells that differed in viability despite being morphologically identical under microscopic observation. SIGNIFICANCE AND IMPACT OF THE STUDY: Flow cytometry has been proved to be a valuable tool for the analysis of sporulation in P. polymyxa cultures, with the unique capacity of distinguishing between endospores and vegetative cells, and between live and dead cells, in the same analysis. An important percentage of non-viable endospores has been found in aged cultures using this method.     

流式细胞术

流式细胞术是一种综合应用光学、机械学、流体力学、电子计算机、细胞生物学、分子免疫学等学科技术,对高速流动的细胞或亚细胞进行快速定量测定和分析的方法。它一秒钟能分析几千个细胞,并同时测定细胞的多个参数,广泛应用于生物医学的许多领域,如测定细胞的特征(形态、膜电位等)和细胞内pH,细胞DNA、蛋白质含量、表面受体、Ca2+等。对生物工程学来说,了解细胞的这些参数尤为重要,因为它们能比用传统技术测得的数据更好地描述细胞群体。从流式细胞仪对细胞多种参数的测定及原理,到它在生物工程学中的应用等方面进行了介绍,并讨论了流式细胞术的局限性和面临的挑战。     

流式细胞术分析和分拣植物染色体

流式细胞术是当染色体、细胞核和细胞等颗粒随着流动的液体（水或缓冲液）通过一个测量点时,被探测器探测到,这样根据颗粒的物理和化学特征而将不同的颗粒分开并计数分拣的技术。流式细胞分析在人类基因组计划中发挥了重要作用,流式细胞技术的应用也适用于植物,目前这个技术应用范围包括流式核型分析,分拣纯化染色体,定位基因,构建文库等。文章综述了流式细胞术在植物基因组分析方面的研究进展。     

Flow cytometry of bacteria: glimpses from the past with a view to the future

Measurement of bacteria and other microorganisms at the level of single cells has progressed enormously over the last couple of decades. Up to the late 1970s, there were no other means than microscopy for observation of single microorganisms, making any type of measurement very cumbersome and tedious, at best. Today, we measure several parameters simultaneously with a precision of a few per cent, and at a rate of 1000 cells per second. The first papers on the use of flow cytometry to measure bacteria appeared only in 1977, although the method had proved highly successful in studies of mammalian cells for almost a decade. There were several reasons for this relatively late introduction, including technical limitations, problems with adequate staining, and, not least, the human factor. Today, flow cytometry has a wide range of microbiological applications, ranging from studies of the bacterial cell cycle and many other cellular characteristics to assessment of antibiotic susceptibility of clinical samples, and monitoring of bacteria and other microorganisms in anything from sewage to sea water. Still, the potential of flow cytometry in microbiology is far from fully utilised. Better instruments and new stains will provide new opportunities to understand, control and exploit this vital part of the biosphere.     

Applications of flow cytometry in plant pathology for genome size determination, detection and physiological status

Flow cytometers are probably the most multipurpose laboratory devices available. They can analyse a vast and very diverse range of cell parameters. This technique has left its mark on cancer, human immunodeficiency virus and immunology research, and is indispensable in routine clinical diagnostics. Flow cytometry (FCM) is also a well-known tool for the detection and physiological status assessment of microorganisms in drinking water, marine environments, food and fermentation processes. However, flow cytometers are seldom used in plant pathology, despite FCM's major advantages as both a detection method and a research tool. Potential uses of FCM include the characterization of genome sizes of fungal and oomycete populations, multiplexed pathogen detection and the monitoring of the viability, culturability and gene expression of plant pathogens, and many others. This review provides an overview of the history, advantages and disadvantages of FCM, and focuses on the current applications and future possibilities of FCM in plant pathology.     

In vivo photothermal flow cytometry: imaging and detection of individual cells in blood and lymph flow

Flow cytometry is a well-established, powerful technique for studying cells in artificial flow in vitro. This review covers a new potential application of this technique for studying normal and abnormal cells in their native condition in blood or lymph flow in vivo. Specifically, the capabilities of the label-free photothermal (PT) technique for detecting and imaging cells in the microvessel network of rat mesentery are analyzed from the point of view of overcoming the problems of flow cytometry in vivo. These problems include, among others, the influences of light scattering and absorption in vessel walls and surrounding tissues, instability of cell velocity, and cells numbers and positions in a vessel's cross-section. The potential applications of this new approach in cell biochemistry and medicine are discussed, including molecular imaging; studying the metabolism and pathogenesis of many diseases at a cellular level; and monitoring and quantifying metastatic and apoptotic cells, and/or their responses to therapeutic interventions (e.g., drug or radiation), in natural biological environments.     

Alveolar macrophage-environmental particle interaction: analysis by flow cytometry

Inhaled particulates such as pollutant particles, allergens, and microorganisms are rapidly cleared by alveolar macrophages (AMs). Methods for analysis of AM-particle interaction have been hindered by the lack of a convenient assay. Flow cytometry offers rapid, sensitive, and reproducible measurements of single cells in suspension. Multiple parameters can be measured in real time. Here we will review the application of flow cytometry to the study and characterization of AM receptors for unopsonized environmental particles. We will discuss the role of this technique in identifying a key AM receptor system involved in lung defense. Multiparametric flow cytometry to analyze intracellular functional parameters, though a powerful and unique tool, needs to be interpreted with caution. We will also discuss the advantages and limitations of flow cytometry in analysis of AM-particle interaction.     

Biofilms and their impact on the food industry

Biofilm could be defined as a complex communities of microorganisms seen affixed to surfaces, they form clusters without sticking to any surface and buried firmly in an extracellular matrix (ECM). This matrix is formed by microorganisms in the formation of either extracellular polymeric substances (EPSS) or extracellular polymer. Many reviews have addressed the negative consequences of biofilm production in the food industry, among which we talk about biofilms being responsible for spoilage microorganisms and foodborne pathogens such as Listeria monocytogenes, Bacillus cereus etc. These contamination could be linked to biofilms presence in the processing plant. Although researches have tried conferring solutions to these challenges in the food industry, however, in this review we have tried to focus on the positive impact of biofilms formed in the food industry. It is critically expedient while trying to find the solution to the challenges of biofilm in the food industry to develop and give a major focus on the advantages and positive impact biofilm has in the food industry, which has been greatly neglected. Hence in this article, we have highlighted some positive impacts of biofilms formed in the food industry, like enhancing plant health and productivity of food products, as an agent of water and wastewater treatment in the food industry, as a tool in reducing the amount of excess sludge in the wastewater treatment plant. The development of edible biofilms, fermented food products and the production of biodegradable food packaging are also part of biofilms beneficial roles in the food industries.     

流式细胞仪的原理、应用及最新进展

流式细胞术是一种采用激光束激发单行流动的细胞,对它的散射光和携带的荧光进行探测,从而完成细胞分析和分选的技术。以流式细胞术为核心技术,流式细胞仪集光学、电子学、生物学、免疫学等多门学科和技术于一体,能够高效分析微小颗粒(如细胞,细菌)的先进科技设备。它对社会产生了深远的影响,成为了科学研究的必要工具。最近几年,流式细胞仪取得了长足进步。为了深入的了解它,本文从流式细胞仪的工作原理和技术指标,在临床医学、生物学、生殖学和制药学中的应用,以及它的世界格局、仪器功能的最新进展三方面,进行了简明、扼要的论述。展望未来:功能专业化、自动化,体积小型化,多色多参数分析能力提高和分析分选速度更快成为流式细胞仪发展的趋势。     

流式细胞术在高等植物研究中的应用

流式细胞术(FCM)是根据所测定的各种细胞性质的不同组合,从细胞群体中把某个亚群分选出来,并对它的功能和形态学进行研究或进一步培养分析。流式细胞术具有快速、灵敏和同时进行多参数检测等优点,对其基本原理和在高等植物中的应用进行了介绍。     

Application of flow cytometry to segregated kinetic modeling based on the physiological states of microorganisms

Flow cytometry (FC) has been introduced to characterize and to assess the physiological states of microorganisms in conjunction with the classical plate-counting method. To show the applicability of the technique, in particular for the development of kinetic models, pure culture fermentation experiments were followed over time, using both prokaryotic (Lactobacillus hilgardii) and eukaryotic (Saccharomyces cerevisiae) microorganisms growing in standard culture media (MRS and YPD). The differences observed between the active and viable cells determined by FC and CFU, respectively, allowed us to determine that a large number of cells were in a viable but nonculturable (VBNC) state, which resulted in a subpopulation much larger than the damaged-cell (double-stained) subpopulation. Finally, the determination of the evolution of viable, the VBNC, and the dead cells allowed us to develop a segregated kinetic model to describe the yeast and the bacteria population dynamics and glucose consumption in batch cultures. This model, more complete than that which is traditionally used, based only on viable cell measurements, describes better the behavior and the functionality of the cultures, giving a deeper knowledge in real time about the status and the course of the bioprocesses.     

Production of precise microbiology standards using flow cytometry and freeze drying.

BACKGROUND: Quality control standards provide a quantity of microorganisms for routine use in microbiology to demonstrate the efficacy of testing methods and culture media. Standards are normally prepared by diluting a culture of microorganisms to obtain a suspension that contains an estimated number of colony-forming units per milliliter. The variability and inaccuracy of these standards increase the potential for false results. Flow cytometry has been used extensively to prepare precise standards of Cryptosporidium and Giardia that contain exact numbers of organisms in a volume of liquid (1). However, the same levels of accuracy have yet to be obtained for bacterial quality control standards. METHODS: A modification of a Becton Dickinson FACScalibur flow cytometer enabled 30 bacterial cells to be sorted into a single droplet, mixed with a cryoprotective solution within the droplet, and frozen in liquid nitrogen. The frozen droplets were then freeze dried for stable preservation of the viable bacterial cells. RESULTS: A freeze-dried sphere 3 mm in diameter was produced, which contained 30 microorganisms. The within-batch variation for these freeze-dried spheres was no greater than two standard deviations, and the between-batch variation was less than one standard deviation. CONCLUSIONS: Bacterial reference controls can now be produced with consistent accuracy and unparalleled precision, thus enabling harmonization across the microbiological testing industry.     

Fountain Flow cytometry, a new technique for the rapid detection and enumeration of microorganisms in aqueous samples.

BACKGROUND: Pathogenic microorganisms are known to cause widespread waterborne disease worldwide. There is an urgent need to develop a technique for the real-time detection of pathogens in environmental samples at low concentrations, <10 microorganisms/ml, in large sample volumes, > or =100 ml. METHODS: A novel method, Fountain Flowtrade mark cytometry, for the rapid and sensitive detection of individual microorganisms in aqueous samples is presented. Each sample is first incubated with a fluorescent label and then passed as a stream in front of a laser, which excites the label. The fluorescence is detected with a CCD imager as the sample flows toward the imager along its optical axis. The feasibility of Fountain Flow cytometry (FFC) is demonstrated by the detection of Escherichia coli labeled with ChemChrome CV6 and SYBR Gold in buffer and natural river water. RESULTS: Detections of labeled E. coli were made in aqueous suspensions with an efficiency of 96% +/- 14% down to a concentration approximately 200 bacteria/ml. CONCLUSIONS: The feasibility of FFC is demonstrated by the detection of E. coli in buffer and natural river water. FFC should apply to the detection of a wide range of pathogenic microorganisms including amoebae.