Statistical analysis and biological interpretation of the flow cytometric heterogeneity observed in bacterial axenic cultures

Histogram comparison and meaningful statistics in flow cytometry is probably the most widely encountered mathematical problem in flow cytometry. Ideally, a test for determining the statistical equality or difference of flow cytometric distributions will identify the significant differences or similarities of the obtained histograms. This situation is of particular interest when flow cytometry is used to study the heterogeneity of axenic bacterial populations. We have statistically measured the heterogeneity of successive cytometric measures, the modifications produced after 20 transfers from the same culture, and the differences between 20 subcultures of identical origin. The heterogeneity of the bacterial populations and the similarity of the obtained 360 histograms were analysed by standard statistical methods. We have studied bacterial axenic cultures in order to detect, quantify and interpret their cytometric heterogeneity, and to assess intrinsic differences and differences produced by laboratory manipulations. We concluded that the standard axenic cultures have a considerable intrinsic cellular and molecular heterogeneity. We suggest that the heterogeneity we have detected basically has two origins: cell size diversity and cell cycle variations.     

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.     

Flow cytometry and cell sorting of heterogeneous microbial populations: the importance of single-cell analyses.

The most fundamental questions such as whether a cell is alive, in the sense of being able to divide or to form a colony, may sometimes be very hard to answer, since even axenic microbial cultures are extremely heterogeneous. Analyses that seek to correlate such things as viability, which is a property of an individual cell, with macroscopic measurements of culture variables such as ATP content, respiratory activity, and so on, must inevitably fail. It is therefore necessary to make physiological measurements on individual cells. Flow cytometry is such a technique, which allows one to analyze cells rapidly and individually and permits the quantitative analysis of microbial heterogeneity. It therefore offers many advantages over conventional measurements for both routine and more exploratory analyses of microbial properties. While the technique has been widely applied to the study of mammalian cells, is use in microbiology has until recently been much more limited, largely because of the smaller size of microbes and the consequently smaller optical signals obtainable from them. Since these technical barriers no longer hold, flow cytometry with appropriate stains has been used for the rapid discrimination and identification of microbial cells, for the rapid assessment of viability and of the heterogeneous distributions of a wealth of other more detailed physiological properties, for the analysis of antimicrobial drug-cell interactions, and for the isolation of high-yielding strains of biotechnological interest. Flow cytometric analyses provide an abundance of multivariate data, and special methods have been devised to exploit these. Ongoing advances mean that modern flow cytometers may now be used by nonspecialists to effect a renaissance in our understanding of microbial heterogeneity.     

Changes in viability and macromolecular content of long-term batch cultures of Salmonella typhimurium measured by flow cytometry

Exposure of many Gram-negative bacteria to prolonged starvation induces alternative programmes of gene expression, along with a transition into a dormant condition sometimes referred to as a viable non-culturable (VBNC) state. Knowledge of how pathogenic species respond to nutrient limitation is therefore important for their detection and dissemination. This study used flow cytometry, coupled with fluorescent dyes for viability and macromolecular content, to study the responses of the pathogen Salmonella typhimurium to prolonged batch culture. Statistical analysis of the flow cytometric data, together with total and culturable cell counts, failed to demonstrate a VBNC state in this pathogen, contrary to reports from other workers. Analysis of rRNA and protein content identified a small proportion of cells in 110 day-old cultures that represented an active sub-population. This observation may provide an explanation for the long-term survival properties of this organism during prolonged exposure to nutrient limitation, as well as the high degree of heterogeneity observed in labelled cells.     

Flow cytometry and bacterial pathogenesis

Our understanding of microbial adaptations to diverse and threatening environments is limited by the assumption that the behavior of individual bacteria can be accurately determined by measuring the behavior of populations. Recent advances in gene expression reporter systems, fluorescence microscopy and flow cytometry allow microbiologists to explore the complex interactions between bacteria and their environment with single cell resolution. The application of these technologies has been particularly useful in systems, such as host-pathogen interactions, where genetic analysis is often cumbersome. Recently, flow cytometry is increasingly being applied to study host-pathogen interactions.     

饮用水微生物的安全快速检测

【目的】为了更好地分析饮用水中的微生物含量。【方法】利用流式细胞术(Flowcytometry,FCM)、ATP测定方法检测瓶装无气饮用水中的微生物数量、可同化有机碳(Assimilable organic carbon,AOC)含量以及微生物活性,并将检测结果与传统的饮用水微生物检测技术相对照。【结果】FCM方法可快速区分水样中的活性细菌和非活性细菌,AOC含量反映了水样中微生物再生能力;而ATP检测方法也能比异养细菌平板计数法(Heterotrophic plate count,HPC)更好地反映瓶装无气饮用水中的实际微生物含量。【结论】FCM、ATP测定方法要明显优于依赖于培养的传统方法。     

On multiparameter data analysis in flow cytometry

Increasing numbers of parameters that are accessible to simultaneous measurement in flow cytometric instruments, combined with the extremely large sample sizes common in flow cytometry, make it necessary to examine methods of multivariate statistics for their applicability to problems of visualization and quantitative analysis of flow cytometric data. This article describes some approaches to dimensionality reduction that appear well suited for data sets obtained by flow cytometry.     

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.     

Cytometry and flow cytometry of 4',6-diamidino-2-phenylindole (DAPI)-stained suspensions of nuclei released from fresh and fixed tissues of plants

Cytometry and flow cytometry were used to study characteristics of fluorescence of the DNA-DAPI complex in nuclei released from different fresh and formaldehyde-fixed pea ( Pisum sativum L. cv. Lincoln) tissues. The two methods of isolation are compared and discussed as well as their possible use for quantitative analysis of DNA in plant tissues. With fixed tissues it is possible to obtain a number of nuclei sufficient for the flow cytometric analysis, even using small amounts of plant tissue.     

Distinction between HLA class I-positive and -negative cervical tumor subpopulations by multiparameter DNA flow cytometry

BACKGROUND: The study of the molecular-genetic basis of heterogeneity of HLA class I expression in solid tumors is hampered by the lack of reliable rapid cell-by-cell isolation techniques. Hence, we studied the applicability of a flow cytometric approach (Corver et al.: Cytometry 2000;39;96-107). METHODS: Cells were isolated from five fresh cervical tumors and simultaneously stained for CD45 or vimentin (fluorescein isothiocyanate fluorescence), Keratin (R-phycoerythrin fluorescence), HLA class I (APC fluorescence), and DNA (propidium iodide fluorescence). A dual-laser flow cytometer was used for fluorescence analysis. Tissue sections from the corresponding tumors were stained for HLA class I antigens, keratin, vimentin, or CD45. RESULTS: Flow cytometry enabled the simultaneous measurement of normal stromal cells (vimentin positive), inflammatory cells (CD45 positive), epithelial cells (keratin positive), and DNA content readily. Normal stromal/inflammatory cells served as intrinsic HLA class I-positive as well as DNA-diploid references. Good DNA histogram quality was obtained (average coefficient of variation < 4%). Intratumor keratin positive subpopulations differing in HLA class I expression as well as DNA content could be clearly identified. Losses of allele-specific HLA class I expression found by immunohistochemistry were also detected by flow cytometry. CONCLUSIONS: We conclude that multiparameter DNA flow cytometry is a powerful tool to study loss of HLA class I expression in human cervical tumors. The method enables flow-sorting of discrete tumor and normal cell subpopulations for further molecular genetic analysis.     

Flow cytometric analysis of microorganisms

The application of flow cytometry to microorganisms is as old as the technique itself, but it has historically been underexploited for microbial applications. This is now being reversed and microbiologists are ideally placed to benefit from recent technological advances. While earlier papers demonstrated the use of flow cytometry for studies of viability and taxonomy, recent developments in bioinformatics and reporter gene technologies are leading to novel applications in microbiology. Variants of green fluorescent protein have been used for the study of conditional microbial gene regulation in medically important host-pathogen interactions and fluorescence-activated cell sorting is being applied to the isolation of novel mutants in directed evolution studies. This paper reviews the reasons for the delay in the application of flow cytometry to microbial problems, the range of applications, and their limitations and considers the progress made in developing new strategies for use in microbiological investigations.     

Toward a self-similar theory of microbial populations

A fresh quest is made of segregated cell models of microbial populations with a view to determine whether the multivarite distribution of physiological states, during transient growth, can attain self-similar forms (i.e., become time invariant) when each physiological state variable is scaled with respect to its population average. Such self-similar growth situations are believed to be more general than those of balanced growth. The conditions under which self-similarity is possible are investigated. Thus conditions are stipulated on the synthesis rates of different physiological entities, cell division rate, and the partitioning of the parent cell's components among the daughter cells (assuming binary division) in order for self-similar growth to be attained. Subject to the attainment of self-similar growth, it is shown that cytometric data can be analyzed systematically to determine how the rates of syntheses of various biochemical entities and cell division rates vary with the physiological entities that are measured. Inverse problems, represented by algebraic systems, are identified which will potentially allow flow cytometric data to be inverted to yield quantitative information on the absolute rates of cellular growth and reproductory processes as a function of the cell states chosen for measurement. It is suggested that the methods become more effective when cytometry can be used to make direct observations on dividing cells so that the number of unknowns in the inverse problem can be reduced, thus facilitating its more complete solution. Preliminary analysis of cytometric data obtained in the literature show promise of self-similarity and thus the possibility of application of the methods discussed here. (c) 1994 John Wiley & Sons, Inc.     

Flow cytometry as a useful tool for process development: rapid evaluation of expression systems.

Flow cytometry is an established tool in fundamental studies of single-cell microbial physiology. Here we show that it can also provide valuable information for process development. Using recombinant Escherichia coli strains, which express the protein-based polymer (GVGIP)(260)GVGVP, the utility of flow cytometry in monitoring and optimization of fermentations is demonstrated. Single cell right angle light scatter was found to be significantly affected by intracellular product formation possibly due to the formation of inclusion bodies. Translational fusions with green fluorescent protein (GFP) enabled monitoring of product accumulation, as well as plasmid free cell fraction (PFCF). Such fusions also allowed rapid evaluation of induction strategies and three different expression systems based on the T7 promoter, T7-lac promoter and the P(BAD) promoter. The expression system based on the P(BAD) promoter was found to be superior to the T7-based system.     

Development of a flow cytometric assay to study glucocorticoid receptor-mediated gene activation in living cells

A flow cytometry-based reporter gene assay was developed and utilized to measure glucocorticoid receptor (GR)-mediated gene activation at the single cell level in living cells. A reporter gene was generated that contains two copies of the glucocorticoid response element and an E1b TATA box upstream of a destabilized enhanced green fluorescent protein. Glucocorticoid activation of the reporter gene in Cos-1 and HTC cell lines was measured in vivo by flow cytometry and was shown to be dose dependent, leading to an increase in total fluorescence of the cell population. Flow cytometric analysis indicated this increase in total fluorescence per sample resulted from an increase in the number of cells expressing the activated green fluorescent protein (GFP) reporter as well as an overall increase in the mean GFP fluorescence within cells. Activation of reporter gene activity was time dependent occurring as early as 1-2h after dexamethasone addition. Activation of the reporter gene was specific as it exhibited different sensitivities to a range of glucocorticoids and activation could be blocked with glucocorticoid receptor antagonists. Coexpression of the coactivator SRC-1a or P65 subunit of NF-kappa B with GR led to enhancement or repression, respectively. Taken together, these data suggest the reporter-based flow cytometry assay is an effective method for analyzing glucocorticoid receptor-mediated gene expression at the single cell level in living cells.     

Flow cytometric analysis of porcine preadipocytes.

In this report, conditions have been established for utilizing monoclonal antibodies and fluorescence activated flow cytometry in studying antigen expression by primary porcine stromal-vascular cells cultured under various conditions. Single cells were isolated from cultures maintained in DME/F12 medium containing 10% fetal bovine serum, 2% pig serum, and containing 2% pig serum and 10 nM dexamethasone supplemented with growth hormone (GH), tumor necrosis factor-alpha (TNF-alpha), and transforming growth factor-beta (TGF-beta). Flow cytometric analyses revealed that the proportion of cells expressing detectable levels of the AD-1 cells surface antigen was greater in cultures supplemented with 2% pig serum and 10 nM dexamethasone than in other media. In cultures, GH, TNF-alpha and TGF-beta each inhibited lipid deposition, whereas TNF-alpha and TGF-beta, but not GH, inhibited AD-1 antigen expression. Inhibition of lipid deposition as well as antigen expression by TNF-alpha and TGF-beta was reversible, but inhibition of cluster formation by GH was not reversed upon removal from cultures. In summary, differential effects of factors on surface antigen expression by preadipocytes are detectable by flow cytometry. Flow cytometric analysis using monoclonal antibodies produced against key developmentally regulated cell surface antigens is potentially a powerful analytical approach to the study of adipocyte development.     

Rapid flow cytometry – Nile red assessment of PHA cellular content and heterogeneity in cultures of Pseudomonas aeruginosa 47T2 (NCIB 40044) grown in waste frying oil

The accumulation of cytoplasmic polyhydroxyalkanoates (PHAs) and the heterogeneity of bacterial populations were analysed by flow cytometry and SYTO-13 and Nile red staining in rhamnolipid-producing Pseudomonas aeruginosa cultures grown in waste frying oil as carbon source. A combination of SYTO-13 and Nile red fluorescence with cytometric forward and side scatter values may allow increases in the final production of polyhydroxyalkanoates (PHA) by two basic mechanisms: (i) rapid assessment of polyhydroxyalkanoate content and (ii) definition of flow cytometric cell sorting protocols to select high polyhydroxyalkanoate (PHA)-producing strains. We report a rapid (less than 30 min) flow cytometric assessment of PHAs in Pseudomonas aeruginosa 47T2 following Nile red staining: (i) to estimate cellular PHAs content; (ii) to study heterogeneity of the batch cultures producing PHAs and (iii) to establish the basis for sorting sub-populations with a high capacity to accumulate PHAs.     

Flow cytometry and cell sorting

Flow cytometry has become an important research tool in cytology, genetics, immunology, and microbiology. The information gained from cytometric instruments is quantitative and of high statistical precision, enabling resolution of cell subpopulations. Although increasing, application to cytology is hindered by inadequate appreciation of the nature of flow cytometry and the information obtained. Many cytologic questions can be reexamined from the perspective of this technology to obtain knowledge not accessible with conventional techniques. A flow cytometer and cell sorter are described. The physical, biochemical, and functional properties measurable by these systems are discussed.     

An efficient and rapid transgenic pollen screening and detection method using flow cytometry

Assaying for transgenic pollen, a major vector of transgene flow, provides valuable information and essential data for the study of gene flow and assessing the effectiveness of transgene containment. Most studies have employed microscopic screening methods or progeny analyses to estimate the frequency of transgenic pollen. However, these methods are time-consuming and laborious when large numbers of pollen grains must be analyzed to look for rare transgenic pollen grains. Thus, there is an urgent need for the development of a simple, rapid, and high throughput analysis method for transgenic pollen analysis. In this study, our objective was to determine the accuracy of using flow cytometry technology for transgenic pollen quantification in practical application where transgenic pollen is not frequent. A suspension of non-transgenic tobacco pollen was spiked with a known amount of verified transgenic tobacco pollen synthesizing low or high amounts of green fluorescent protein (GFP). The flow cytometric method detected approximately 75% and 100% of pollen grains synthesizing low and high amounts of GFP, respectively. The method is rapid, as it is able to count 5000 pollen grains per minute-long run. Our data indicate that this flow cytometric method is useful to study gene flow and assessment of transgene containment.     

Sensitive analysis of recombination activity using integrated cell surface reporter substrates.

BACKGROUND:Recombination processes play a crucial role in the functioning of the immune system and are also involved in mutation events that result in various malignancies. So far the study of recombination activity has frequently relied on the use of reporter substrates that are limited by low sensitivity as well as tedious and distorting readout procedures. METHODS:Immunoglobulin class switch recombination substrates were generated which, upon recombination, resulted in the surface expression of human CD4 or murine MHC class I H-2K(k) and thus allowed for cytometric evaluation. RESULTS:Recombining cells harboring integrated reporter substrates were analyzed by immunofluorescence and flow cytometry and could easily be isolated by high-gradient magnetic cell sorting (MACS). The analysis was not influenced by cloning efficiencies, as would be the case after drug selection, or prokaryotic recombination that might occur after analysis of recovered substrates in bacteria. In addition, cytometric readout is much faster, as it can be performed immediately after recombination. The substrate exhibited properties compatible with the detection of immunoglobulin class switch recombination and permitted the detection of recombination events down to 10(-5) per cell and generation. CONCLUSIONS:The high sensitivity of this system allows precise detection of very rare recombination events and thus permits the study of cell types with extremely low recombination activities.