Differences in stationary-phase cells of a commercial Saccharomyces cerevisiae wine yeast grown in aerobic and microaerophilic batch cultures assessed by electric particle analysis, light diffraction and flow cytometry.

We applied electric particle analysis, light diffraction and flow cytometry to obtain information on the morphological changes during the stationary phase of Saccharomyces cerevisiae. The reported analyses of S. cerevisiae populations were obtained under two different conditions, aerobic and microaerophilic, at 27°C. The samples analysed were taken at between 20 and 50 h from the beginning of culture. To assist in the interpretation of the observed distributions a complexity index was used. The aerobically grown culture reached significantly greater cell density. Under these conditions, the cell density experienced a much lower reduction (3%) compared with the microaerophilic conditions (30%). Under aerobic conditions, the mean cell size determined by both electric particle analysis and light diffraction was lower and remained similar throughout the experiment. Under microaerophilic conditions, the mean cell size determined by electric particle analysis decreased slightly as the culture progressed through the stationary phase. Forward and side scatter distributions revealed two cell subpopulations under both growth conditions. However, in the aerobic growing culture the two subpopulations were more separated and hence easier to distinguish. The distributions obtained with the three experimental techniques were analysed using the complexity index. This analysis suggested that a complexity index is a good descriptor of the changes that take place in a yeast population in the stationary phase, and that it aids in the discussion and understanding of the implications of these distributions obtained by these experimental techniques.

Rapid G+ count and subpopulation assessment of the intestinal bacteria in Apodemus sylvaticus and Mus musculus by flow cytometry.

We report a novel application of calcein-acetomethyl ester in flow cytometry for rapid estimation of the number of G+-bacteria in rodent feces (Apodemus sylvaticus and Mus sp.f. muridae). We also use the combined application of flow cytometry and Syto-13 or Sypro Orange staining to count rapidly the total bacterial population and to describe bacterial subpopulations in the intestine.

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.

Involvement of RNA and DNA in the staining of Escherichia coli by SYTO 13.

AIMS: To assess the extent to which DNA and RNA bacterial content contributes to fluorescent response of SYTO 13. METHODS AND RESULTS: RNA and DNA of Escherichia coli 536 cells were extracted and fluorimetrically quantified to compare the different contents, throughout a 24 h culture, with their SYTO 13 fluorescence emission when analysed by the cytometer. SYTO 13 fluorescence varied depending on the stage of bacterial growth and in accordance with both DNA and RNA content. RNA content accounted for at least two-thirds of the total fluorescence of a cell. Escherichia coli cells were treated with chloramphenicol to improve their RNA content. With this treatment, both nucleic acids remained constant but there was a clear improvement in fluorescent emission. SYTO 13 fluorescence was also studied in E. coli X-1488 minicells. CONCLUSIONS: Although both nucleic acids are implicated, RNA accounts for a major part of SYTO 13 fluorescence. The fluorescence cannot be considered as a direct reflection of nucleic acid content. Other factors, such as topology or supercoiling, need to be considered. SIGNIFICANCE AND IMPACT OF THE STUDY: The results confirm the efficacy of SYTO 13 for labelling bacteria and for assessing the distinct physiological status. A better knowledge of the parameters implicated in its fluorescence emission has been achieved.

Effects of deflected droplet electrostatic cell sorting on the viability and exoproteolytic activity of bacterial cultures and marine bacterioplankton.

The cell-sorting capability of flow cytometers makes it possible to isolate specific populations of cells with pre-defined cytometric characteristics. A better knowledge of the biological effects of the sorting process is necessary for the future cell sorting applications. In this paper we report the effects of flow cytometric sorting on bacterial viability and exoproteolytic activity (EPA) of bacterial cultures and marine bacterioplankton. Sorting bacterial cultures and bacterioplankton samples reduce viability as assessed by plate counts and produce variations in the exoproteolytic activity. These effects indicate that deflected electrostatic sorting may significantly alter the biological properties of the sorted bacteria.

Fate and effect of monoalkyl quaternary ammonium surfactants in the aquatic environment.

The effect of the alkyl chain of quaternary ammonium-based surfactants on their aquatic toxicity and aerobic biodegradability has been studied. Two families of monoalkylquats surfactants were selected: alkyl trimethyl ammonium and alkyl benzyl dimethyl ammonium halides. Acute toxicity tests on Daphnia magna and Photobacterium phosphoreum were carried out and EC50 values in the range of 0.1-1 mg/l were obtained for the two series of cationic surfactants. Although the substitution of a benzyl group for a methyl group increases the toxicity, an incremental difference in toxicity between homologs of different chain length were not observed. Biodegradability of the different homologs was determined not only in standard conditions but also in coastal water, both tests yielding similar results. An increase in the alkyl chain length or the substitution of a benzyl group for a methyl group reduces the biodegradation rate. The degradation of these compounds in coastal waters was associated with an increase in bacterioplankton density, suggesting that the degradation takes place because the compound is used as a growth substrate.

Microbial community dynamics in Mediterranean nutrient-enriched seawater mesocosms: changes in the genetic diversity of bacterial populations.

A mesocosm experiment was performed to study the influence of nutrients on activity and diversity of bacterial assemblages from the Mediterranean Sea. Changes in the diversity of the predominant bacterial populations were monitored by DGGE fingerprinting of PCR products derived from 16S rRNA encoding genes. Fluctuations in the diversity of the most active populations was inferred by performing the DGGE fingerprinting on the basis of the cellular rRNA after reverse transcription and PCR amplification. DNA-derived DGGE patterns obtained from duplicate control and nutrient-enriched mesocosms showed differences in the development of the bacterial communities between control and nutrient-enriched experimental mesocosms. Multidimensional scaling analysis of the DNA-derived DGGE fingerprints indicated that duplicate treatments were reproducible. DNA- and RNA-derived DGGE fingerprints of bacterial assemblages changed over time, showing that the composition of the bacterial assemblages, as well as the most active bacterial populations changed during different phases of the incubation. Sequences of predominant DGGE bands in RNA-derived patterns were similar to 16S rRNA gene sequences of members of the alpha-, gamma- and delta-Proteobacteria and of the Cytophaga-Flavobacterium-Bacteroides phylum (CFB). Bands corresponding to Ruegeria-like bacteria and members of the CFB became especially dominant during the course of incubation, suggesting that these populations were important contributors to bacterial production and activity in the post-grazing phase of the experiment.

Microbial community dynamics in Mediterranean nutrient-enriched seawater mesocosms: changes in abundances, activity and composition.

Quantitative and qualitative changes in bacterial communities from the Mediterranean Sea were compared in duplicate batch mesocosms with or without addition of inorganic nutrients. Methods including traditional microbial ecology techniques, molecular biology and flow cytometry were combined to determine abundances, production, cell size, activity, culturability and taxonomic diversity of bacterial cells. Addition of nutrients and confinement resulted in an increase of bacterial densities which were rapidly controlled by protozoan grazing. Changes in bacterial activity and morphology were observed during the growth phase of bacteria and under grazing pressure. The proportion of medium-size and culturable cells increased during the growth phase. These cells were preferentially consumed by grazers resulting in a strong limitation of bacterial production. As a consequence of the grazing pressure, large cells were produced and contributed to the remaining bacterial productivity after grazing. Grazing had an effect on the taxonomic composition of bacterial communities by preferentially eliminating gamma-Proteobacteria, alpha-Proteobacteria were preserved. It seems that some species from the genera Ruegeria and Cytophaga may have developed defence strategies to escape predation.

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 ofbacterial 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.

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.

Flow cytometric analysis of a marine LAS-degrading consortia.

The specific nucleic acid fluorochrome SYTO-13 was used in flow cytometric analysis to assess changes in the density and heterogeneity of marine bacterial populations which biodegrade linear alkylbenzene sulphonate (LAS). Seawater samples with LAS and incubated in the laboratory (20 degrees C, 100 rpm, 30 days) were used to monitor LAS-degrading consortia. Flow cytometric studies and culture methods were used to characterize the LAS degrading bacterioplankton consortia. Fluorescence and scatter signals enabled us to define three regions (R1, R2 and R3) in the dual parameter cytograms. The distribution of the bacterial counts in these regions allowed us to monitor the formation and evolution of the consortia.

Second-order functions are the simplest correlations between flow cytometric light scatter and bacterial diameter.

Second-order mathematical relationships between bacterial cell diameter determined by electric particle analyser and flow cytometric forward light scatter in axenic cultures are obtained and discussed. Since it is technically impossible today to obtain both measurements for each individual cell, standard regression techniques cannot be applied. To overcome this limitation, we assume that these two parameters are related by a monotone increasing function that enables their mathematical relationships to be studied. Our conclusion is that forward light scatter data cannot be linearly transformed into bacterial size values by an accurate and universal function. However, second-order relationships seem to be the simplest satisfactory relationships between cell diameter and forward light scatter in eubacteria.

Use of nucleic acid dyes SYTO-13, TOTO-1, and YOYO-1 in the study of Escherichia coli and marine prokaryotic populations by flow cytometry.

Three nucleic acid dyes (SYTO-13, TOTO-1, and YOYO-1) were tested on cultures of Escherichia coli and marine prokaryote populations. These dyes stain the RNA and DNA in E. coli but only respond to DNA in marine populations, according to the histograms obtained after DNase and RNase treatments.

Assessment of the effects of gramicidin, formaldehyde, and surfactants on Escherichia coli by flow cytometry using nucleic acid and membrane potential dyes.

Two membrane potential sensitive dyes (Rhodamine 123 and bis-oxonol) and three nucleic acid dyes (propidium iodide, SYTO-13, and SYTO-17) were used to assess the effect of surfactants on Escherichia coli. The ability of E. coli to be stained by these probes was validated at different physiological states. Propidium iodide was used to assess the integrity of cell envelopes. Two double staining methods based on propidium iodide with SYTO-13 and bis-oxonol with SYTO-17 were used to improve the discrimination between bacteria and micelles or aggregated particles generated by the presence of surfactants. A rapid (1 h contact time between cells and surfactants, and less than 5 min for staining and obtaining data) Rhodamine 123 flow cytometric assay was developed to assess the bactericidal effect of surfactants.

Exogenous isolation of Hgr plasmids from coastal Mediterranean waters and their effect on growth and survival of Escherichia coli in sea water.

Four self-conjugative plasmids conferring mercury resistance were exogenously isolated from coastal Mediterranean sea water. All the plasmids isolated (93.2, 73.1, 65.2 and 221 kb) showed narrow-spectrum mercury resistance, and no UV resistance; three of them conferred cadmium resistance (Cdr 1.5 mM) and two of them encoded streptomycin resistance (50 micrograms/ml). Their influence on the survival of the recipient strain Escherichia coli JGL1 in different seawater media was studied and compared with that of two plasmids previously isolated by the same procedure from an epilithic community of the Bristol Channel, Great Britain. A significant negative influence was observed for the largest (221 kb) of the plasmids isolated and one of the epilithic plasmids (239 kb). The effect of the plasmids on E.coli JGL1 was dependent on the seawater media.

Assessment of E. coli and Salmonella viability and starvation by confocal laser microscopy and flow cytometry using rhodamine 123, DiBAC4(3), propidium iodide, and CTC.

Assessment of cell viability using methods which do not require cell culture is essential in the field of aquatic microbiology, since many bacteria known to be present in aquatic environments cannot be grown in culture. The study of bacterial biofilms, which previously needed an epifluorescent microscope, has recently been enhanced by the use of flow cytometry and confocal scanning laser microscopy (CSLM). A method based on the combination of several membrane potential related dyes, a membrane integrity dye and a redox probe was used to measure cell viability by flow cytometry and confocal laser microscopy. Rhodamine-propidium iodide (PI) double staining was used to discriminate viable from nonviable cells in CSLM observations. Membrane depolarization during E. coli and Salmonella starvation measured by DiBAC4(3) incorporation (flow cytometry and CSLM) was found to be in concordance with respiratory activity as detected by a tetrazolium salt (CTC) reduction.

Statistical model fitting Escherichia coli survival data during starvation in artificial sea water.

The survival of Escherichia coli in artificial sea water was studied using linear and nonlinear regression analysis in order to fit the simplest model. The surviving population was evaluated in a rich medium, and two mathematical models, monophase and diphase linear, were considered. The diphase model gave the best fit, although both models can be adjusted to the results obtained. The criteria applied to decide which model was more statistically significant, were residuals, the residual sum of squares and the coefficient of determination. These models are discussed in the light of present day knowledge on survival.

Flow cytometric assessment of Escherichia coli and Salmonella typhimurium starvation-survival in seawater using rhodamine 123, propidium iodide, and oxonol.

The use of flow cytometry in microbiology allows rapid characterization of cells from a nonhomogeneous population. A method based on flow cytometry to assess the effects of lethal agents and the bacterial survival in starved cultures through the use of membrane potential-sensitive dyes and a nucleic acid marker is presented. The use of propidium iodide, rhodamine, and oxonol has facilitated the differentiation of cells of Escherichia coli and Salmonella typhimurium of various states of vitality following various treatments (heat, sonication, electroporation, and incubation with gramicidin) and during starvation in artificial seawater. The fluorescence intensity is directly correlated with viable cell counts for rhodamine 123 labelling, whereas oxonol and propidium iodide labelling is inversely correlated with viable counts. The distribution of rhodamine and oxonol uptake during starvation-survival clearly indicates that single-species starved bacteria are heterogeneous populations, and flow cytometry can be a fundamental tool for quantifying this heterogeneity.

Assessment of survival during starvation of Escherichia coli and Klebsiella pneumoniae in artificial urine: analysis of the kinetics of colony formation.

A kinetic model of colony formation was proposed by Hattori, based on a count of the colonies that appear on a plate in successive short intervals of time. In this model, three parameters (lambda, tr and N infinity) are defined, which reflect the ability of a bacterium to yield colonies and allow us to described the dynamics of bacterial populations in soil and of E. coli at different growth phases. In this paper we report a reparametrization of the kinetic model of colony formation, with the aim of facilitating more accurate calculation of lambda and tr. Moreover, we observed that during the starvation of E. coli and K. pneumoniae in urine, lambda can be used to assess survival, since this parameter clearly decreases during starvation. Retardation time values (tr) were similar in E. coli and K. pneumoniae throughout the starvation experimental period.

Variations in flow cytometric forward scatter signals and cell size in batch cultures of Escherichia coli.

Flow cytometry was used to study the lag, exponential, stationary and death phases of non-fixed cultures of Escherichia coli. Fluctuations in the forward angle scatter signal (FALS) were compared with cell size as measured by scanning electron microscopy at low temperature and image analysis. A correlation between FALS and cell size was not observed, although a correlation (r = -0.8) was obtained between FALS and the age of the culture for the first eleven days of incubation. Marked increases in FALS were observed during the lag phase, which were attributed both to changes in size and changes in structure or chemical composition. The distribution of FALS for all culture phases was asymmetric, and was associated with the cell size distribution.