Flow cytometric identification of microorganisms by dual staining with FITC and PI.

The identification of microorganisms by flow cytometry was evaluated by using a double staining technique with propidium iodide and fluorescein isothiocyanate and a two dimensional analysis. A diverse group of 19 different species and strains of microorganisms was tested to determine if they could be differentiated by flow cytometry. The organisms tested displayed characteristic and distinct two dimensional fluorescent patterns which allowed ready grouping and differentiation into subsets of organisms. The slopes and correlation coefficients of the histograms and the ratio of red to green signals expressed these differences quantitatively and allowed organisms to be placed into one of three groups based on these values. In some instances, as with Streptococcus pneumoniae and pyogenes and Staphylococcus aureus and epidermidis, it was possible to distinguish between species of bacteria from the same genus. The use of dual dye labeling and flow cytometry provided a rapid method of identifying selected microorganisms and may be broadly applicable for the detection and identification of many bacteria and fungi.     

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.     

Vigour, vitality and viability of microorganisms

Abstract Assessment of the vigour, vitality or viability of microorganisms must be done on an individual basis and thus requires the non-invasive interrogation of single organisms. For suspended organisms, flow cytometry provides a powerful means of measurement of a wide range of characteristics. Similar information for microbes in aggregates or growing on surfaces may be obtained by use of confocal scanning laser microscopy. For instance, membrane potential-sensitive fluorophores can distinguish between vigorous, frail and dead cells.     

Single‐Cell Sorting of Microorganisms by Flow or Slide‐Based (Including Laser Scanning) Cytometry

In applied microbiology, strain improvement of microorganisms by conventional selection culture is not always successful, so single‐cell selection of viable cells with the desired characteristics from a large heterogeneous population may be used instead. Single‐cell selection with use of a micromanipulator is possible, but laborious. For many applications, the process has been automated. In this review, an automated method, laser scanning cytometry (LSC), is outlined together with flow cytometry (FCM). FCM is familiar to many microbiologists, but LSC is a microscopic‐slide‐based method that is less well known. One of its advantages is its possible use in the examination of small cell populations.In addition, individual cells can be examined repeatedly, measured automatically and later observed microscopically by the operator, and finally stored (if desired)on the microscopic slide on which they are placed. Fluorescent and other probes are available in abundance for FCM, and almost all might be used in LSC. A number of applications of these methods are cited from the extensive literature (mostly about FCM), but the list of possible applications in this review is far from being exhaustive. This review is intended as an introduction for the applied microbiologist to the manifold uses of LSC.     

When Phase Contrast Fails: ChainTracer and NucTracer,Two ImageJ Methods for Semi-Automated Single Cell Analysis Using Membrane or DNA Staining

Within bacterial populations, genetically identical cells often behave differently. Single-cell measurement methods are required to observe this heterogeneity. Flow cytometry and fluorescence light microscopy are the primary methods to do this. However, flow cytometry requires reasonably strong fluorescence signals and is impractical when bacteria grow in cell chains. Therefore fluorescence light microscopy is often used to measure population heterogeneity in bacteria. Automatic microscopy image analysis programs typically use phase contrast images to identify cells. However, many bacteria divide by forming a cross-wall that is not detectable by phase contrast. We have developed ‘ChainTracer’, a method based on the ImageJ plugin ObjectJ. It can automatically identify individual cells stained by fluorescent membrane dyes, and measure fluorescence intensity, chain length, cell length, and cell diameter. As a complementary analysis method we developed ''NucTracer'', which uses DAPI stained nucleoids as a proxy for single cells. The latter method is especially useful when dealing with crowded images. The methods were tested with Bacillus subtilis and Lactococcus lactis cells expressing a GFP-reporter. In conclusion, ChainTracer and NucTracer are useful single cell measurement methods when bacterial cells are difficult to distinguish with phase contrast.     

Polyphasic detection of cyanobacteria in terrestrial biofilms

Cyanobacterial populations detected on buildings by traditional methods are mainly filamentous, whereas direct microscopy shows that they are principally coccoid morphotypes that often cannot be isolated in culture, but may grow on artificial media when the spatial biofilm relationships are maintained. The polyphasic strategy described here was to select morphologically distinct colonies from rehydrated biofilms for direct DNA amplification, allowing uncultured organisms to be sequenced and their morphology to be characterized by microscopy. DNA data banks currently contain many entries for cyanobacteria of unrecorded morphology, which does not facilitate identification, although genetic variability in a population may be assessed. The sequence homologies of the present biofilm organisms (EMBL accession numbers AJ619681 to 619690) with those in DNA databanks were low, indicating differences between xerophytic cyanobacteria on walls and aquatic species comprising the majority in the databases. Further development of databases for the populations found in this environment, subject to temperature extremes, repeated desiccation and high UV and salt levels, is required.     

Polyphasic Detection of Cyanobacteria in Terrestrial Biofilms

Cyanobacterial populations detected on buildings by traditional methods are mainly filamentous, whereas direct microscopy shows that they are principally coccoid morphotypes that often cannot be isolated in culture, but may grow on artificial media when the spatial biofilm relationships are maintained. The polyphasic strategy described here was to select morphologically distinct colonies from rehydrated biofilms for direct DNA amplification, allowing uncultured organisms to be sequenced and their morphology to be characterized by microscopy. DNA data banks currently contain many entries for cyanobacteria of unrecorded morphology, which does not facilitate identification, although genetic variability in a population may be assessed. The sequence homologies of the present biofilm organisms (EMBL accession numbers AJ619681 to 619690) with those in DNA databanks were low, indicating differences between xerophytic cyanobacteria on walls and aquatic species comprising the majority in the databases. Further development of databases for the populations found in this environment, subject to temperature extremes, repeated desiccation and high UV and salt levels, is required.     

Quantification of the presence and activity of specific microorganisms in nature

Traditional techniques for assessment of microbial numbers and activity generally lack the specificity required for risk assessment following environmental release of genetically engineered microbial inocula. Immunological and molecular-based techniques, such as DNA probing and genetic tagging, were initially used to determine the presence or absence of microorganisms in environmental samples. Increasingly they are being developed for quantification of populations of specific organisms, either indigenous or introduced, in the environment. In addition, they are being used to quantify the activity of particular organisms or groups of organisms, greatly extending the range of techniques available to the microbial ecologist. This article reviews the use of traditional techniques for the quantification of microbial population size and activity and the application of molecular techniques, including DNA probing, genetic marking, use of fluorescent probes, and quantitative PCR, in combination with advanced cell detection techniques such as confocal laser scanning microscopy and flow cytometry.     

肠道微生物培养的研究进展及应用

肠道中栖居着组成复杂、功能多样的微生物群,这些微生物群在宿主免疫、营养吸收、代谢调节等方面发挥着重要作用。随着测序技术的快速发展,肠道微生物研究通过16S rRNA基因测序和宏基因组测序产生了大量的数据,其中许多未组装的序列成为微生物“暗物质”。近年来,不少研究利用多种不同微生物分离培养方法,结合高通量鉴定技术,从人、小鼠、猪肠道中分离了大量的微生物,丰富了菌株资源,为解析微生物“暗物质”以及后续肠道微生物功能和应用研究提供了基础和保障。尽管微生物的可培养性受到多种因素的影响,大部分微生物尚处于“未培养”的状态,但无论是病因研究还是生理和遗传特征的解析都离不开微生物实体资源的获取。肠道微生物的分离培养对微生物研究从关联分析向菌群功能验证、因果机制解析和功能菌株开发的深入研究具有重要意义。本文旨在探讨和综述影响微生物可培养性的因素,总结回顾肠道微生物的培养方法并阐述肠道微生物培养研究的进展,以期为肠道微生物培养研究提供新的视角。     

Indicators of Pneumocystis carinii viability in short-term cell culture.

Growth of P. carinii in culture has been difficult to document in the absence of reliable methods for distinguishing live from dead organisms. We studied three markers of cell function in P. carinii during the course of short-term cell culture, and correlated these with the number of P. carinii present in culture supernatants. The markers were glucan synthase activity, esterase activity and cell membrane integrity. The last two were assessed by double staining with fluorescein diacetate and propidium iodide followed by analysis of fluorescence using flow cytometry. The rise in P. carinii number after 5 to 7 days in culture was associated with increased glucan synthase activity. Flow cytometry analysis of day-6 P. carinii cultures confirmed that over 80% of the organisms catalyzed the conversion of fluorescein diacetate to fluorescein and excluded propidium iodide. The demonstration of three indices of metabolic activity in an expanding P. carinii population has confirmed the efficacy of a culture system as a means of sustaining the continued activity, albeit short-lived, of viable P. carinii.     

Enrichment of slow-growing marine microorganisms from mixed cultures using gel microdrop (GMD) growth assay and fluorescence-activated cell sorting

Encapsulation of cells in agarose gel microdrops (GMDs) combined with fluorescence-activated cell sorting (FACS) has been used previously to analyze and recover specific mammalian, bacterial, and yeast cell populations. Recently, we have developed a method to enrich mixed bacterial populations for slow-growing microorganisms using the GMD Growth Assay combined with fluorochrome staining and flow cytometry. Here, we demonstrate the feasibility of using this experimental approach to detect clonogenic growth of individual bacteria within GMDs in less than 3 h and to separate subpopulations based on differential growth rates. We show that after sorting, organisms remain viable and can be propagated in culture for further analysis.     

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.     

Heterogeneity of stress gene expression and stress resistance among individual cells of Saccharomyces cerevisiae

Knowledge of gene expression and cellular responses in microorganisms is derived from analyses of populations consisting of millions of cells. Analytical techniques that provide data as population averages fail to inform of culture heterogeneity. Flow cytometry and fluorescence techniques were used to provide information on the heterogeneity of stress-responsive gene expression and stress tolerance in individual cells within populations. A sequence of DNA encoding the heat shock and stress response elements of the Saccharomyces cerevisiae HSP104 gene was used to express enhanced green fluorescent protein (EGFP). When integrated into the genome of yeast strain W303-1A, intrinsic expression of EGFP increased about twofold as cells progressed from growth on glucose to ethanol utilization in aerobic batch cultures. Staining of cells with orange/red fluorescent propidium iodide (PI), which only enters cells that have compromised membrane integrity, revealed that the population became more tolerant to 52 degrees C heat stress as it progressed from growth on glucose and through the ethanol utilization phase of aerobic batch culture. Exposure of cultures growing on glucose to a mild heat shock (shift from 25 degrees C to 37 degrees C) resulted in significantly increased expression of EGFP in the population. However, there was heterogeneity in the intensity of fluorescence of individual cells from heat-shocked cultures, indicating variability in the strength of stress response in the clonal population. Detailed analysis of the heterogeneity showed a clear positive trend between intensity of stress response and individual cell resistance, measured in terms of PI exclusion, to heat stress at 52 degrees C. Further experiments indicated that, although the mean gene expression by a population is influenced by the genetic background, the heterogeneity among individual cells in clonal populations is largely physiologically based.     

Evaluation of the cell growth of mycobacteria using Mycobacterium smegmatis mc2 155 as a representative species

The study of the in vitro cell growth of mycobacteria still remains a fastidious, difficult, and time-consuming procedure. In addition, assessing mycobacterial growth in the laboratory is often complicated by cell aggregation and slow growth-rate. We now report that the use of a stainless steel spring in the culture led to an absence of large cell clumps, to a decrease of dead cells in the exponential phase and to growth of a more homogeneous population of large cells. We also report that flow cytometry is a rapid, simple and reliable approach to monitor mycobacterial cell growth and viability. Here, we monitored Mycobacterium smegmatis cellular growth by optical density, dry cell mass, and colony forming units; in addition, viability, cell size and granularity profiles were analyzed by flow cytometry, and cell morphology by electron microscopy. Cultures monitored by flow cytometry may lead to a better understanding of the physiology of mycobacteria. Moreover, this methodology may aid in characterizing the cell growth of other fastidious species of microorganisms.     

Ecophysiology and breeding of mycoparasitic Trichoderma strains (a review)

Losses due to plant diseases may be as high as 10-20% of the total worldwide food production every year, resulting in economic losses amounting to many billions of dollars and diminished food supplies. Chemical control involves the use of chemical pesticides to eradicate or reduce the populations of pathogens or to protect the plants from infection by pathogens. For some diseases chemical control is very effective, but it is often non-specific in its effects, killing beneficial organisms as well as pathogens, and it may have undesirable health, safety, and environmental risks. Biological control involves the use of one or more biological organisms to control the pathogens or diseases. Biological control is more specialized and uses specific microorganisms that attack or interfere with the pathogens. The members of the genus Trichoderma are very promising against soil-born plant parasitic fungi. These filamentous fungi are very widespread in nature, with high population densities in soils and plant litters [1]. They are saprophytic, quickly growing and easy to culture and they can produce large amounts of conidia with long lifetime.     

Expanding the known diversity and environmental distribution of cultured and uncultured bacteria

Microorganisms represent the largest component of biodiversity in our biosphere. Traditional methods of bacterial identification depend on their culture on laboratory media and the comparison of their phenotypic characteristics. They include cellular morphology, motility, staining reactions of cell walls, ability to grow on different media and biochemical tests. These methods have many limitations and only a very small fraction of microorganisms have been cultivated. To date, molecular methods based on 16S rRNA sequences and their phylogenetic analysis are widely used for reliable identification, particularly for hard-to-culture microbial pathogens. These so-called < molecular methods > do not require laboratory culture of isolated organisms, and many novel non-described phyla have been detected, improving our view of bacterial diversity. Novel strategies for culturing the < uncultivated > are now under development, which are leading to the complete characterization of these new bacteria. More recently, meta- or ecogenomics, based on the complete sequencing of clones containing cosmids or bacterial artificial chromosomes with inserts, addresses the genetic potential of a sample irrespective of whether the microorganisms can be cultured or not. This has considerably extended our view of microbial diversity at the genomic level and the probability of finding new genes and their products suitable for the biotechnological and pharmaceutical industry.     

Fitness effects of mutations in bacteria

Mutation is the primary source of variation in any organism. Without it, natural selection cannot operate and organisms cannot adapt to novel environments. Mutation is also generally a source of defect: many mutations are not neutral but cause fitness decreases in the organisms where they arise. In bacteria, another important source of variation is horizontal gene transfer. This source of variation can also cause beneficial or deleterious effects. Determining the distribution of fitness effects of mutations in different environments and genetic backgrounds is an active research field. In bacteria, knowledge of these distributions is key for understanding important traits. For example, for determining the dynamics of microorganisms with a high genomic mutation rate (mutators), and for understanding the evolution of antibiotic resistance, and the emergence of pathogenic traits. All of these characteristics are extremely relevant for human health both at the individual and population levels. Experimental evolution has been a valuable tool to address these questions. Here, we review some of the important findings of mutation effects in bacteria revealed through laboratory experiments.     

Isolation of Extracellular Microvesicles from Cell Culture Medium: Comparative Evaluation of Methods

Extracellular vesicles (EV) are secreted by cells of multicellular organisms. EV mediate specific mode of intercellular communication by “horizontal” exchange of substances and information. This phenomenon seems to have an essential biological significance and became a subject of intensive research. Biogenesis, structural and functional EV features are usually studied in vitro. Several methods of EV isolation from cell culture medium are currently used; however, selection of a particular method may have a significant impact on obtained results. The choice of the optimal method is usually determined by the amount of starting biomaterial and the aims of the research. We have performed a comparative analysis of four different methods of EV isolation from cell culture medium: differential ultracentrifugation, ultracentrifugation with 30% sucrose/D₂O “cushion,” precipitation with plant proteins and latex-based immunoaffinity capturing. EV isolated from several human glial cell lines by different approaches were compared in terms of the following parameters: size, concentration, EV morphology, contamination by non-vesicular particles, content of exosomal tetraspanins on the EV surface, content of total proteins, total RNA, and several glioma-associated miRNAs. The applied methods included nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), cryo-electron microscopy, flow cytometry and RT-qPCR. Based on the obtained results, we have developed practical recommendations that may help researchers to make the best choice of the EV isolation method.     

Large Bodies of Mycoplasma and L-Form Organisms

The large bodies of various Mycoplasma and L-form organisms were studied by ultraviolet fluorescence microscopy of preparations stained with various fluorochromes. Primuline and Thioflavine S specifically stained the outer portion or rim of the large bodies, and the fluorescence characteristics of the stained bodies differed from those for other microorganisms and for spheroplasts and protoplasts. Small granular structures similar in size and morphology to minimal reproductive units were observed within some of the large bodies by phase microscopy and by fluorescence microscopy with acridine orange or Coriphosphine O. Micromanipulation probing of the large bodies revealed their elastic nature; many of the large bodies could be subdivided into two or more smaller circular bodies, each retaining the fluorescence staining properties of the parent body. Under these conditions, however, a few of the large bodies were ruptured, leaving the stainable outer boundary area as a stable residual structure. The large bodies were somewhat resistant to various rigorous treatments normally employed to eliminate viability of Mycoplasma and L-form cultures. Structures similr to large bodies were observed in various natural tissues, and structures resembling large bodies in size, morphology, fluorescence staining characteristics, and reaction to micromanipulation probing were reconstructed from an acetone extract of egg yolk. Overall, the large bodies of Mycoplasma and L-form organisms appeared to be structures resulting from accumulations of metabolic by-products and medium components within or on which minimal reproductive units had become entrapped, although it could not be ruled out that they might be defined structures specifically formed during culture as protective lipoidal sacs for the minimal reproductive units.     

Biological and morphological characterization of human neonatal fibroblast cell culture B-HNF-1

In the present study, human neonatal fibroblasts were isolated from a two-month-old human male. The purpose of the present investigation was the analysis of the morphology (light and transmission electron microscopy), karyotype and growth characteristics of the human neonatal fibroblast cell culture B-HNF-1. Moreover, STR typing and mitochondrial DNA amplification and sequencing was also performed. Analysis of chromosomes count showed that B-HNF-1 cell culture is diploid and has normal male karyotype 46, XY, which was stable during cultivation. The transmission electron microscopy demonstrated the ultra-structure of the B-HNF-1 cells; they have typical morphological features of proteosynthesis-active cells. Large number of fibroblasts bearing different shapes and surface characteristics adhered to the substrate with microvilli and filopodia. Our in vitro expanded fibroblasts have a large and irregular nucleus with prevalence of euchromatin. One to three nucleoli are present in each nucleus. The cytoplasm contains a richly developed rough endoplasmic reticulum and prominent Golgi apparatus cisterns. The result of the fragment analysis is a DNA profile defined as multiplex of STR markers and sex determining system. Sequencing analysis of hypervariable segments of control region of mitochondrial DNA showed haplotype that belongs to haplogroup W. In this study, we complexly characterized a new cell culture of human neonatal fibroblasts B-HNF-1 from different points of view. This culture should be used for further biomedical experiments.