Hazel M. Davey

Life, Death, and In-Between: Meanings and Methods in Microbiology

ABSTRACT Determination of microbial viability by the plate count method is routine in microbiology laboratories worldwide. However, limitations of the technique, particularly with respect to environmental microorganisms, are widely recognized. Many alternatives based upon viability staining have been proposed, and these are often combined with techniques such as image analysis and flow cytometry. The plethora of choices, however, adds to confusion when selecting a method. Commercial staining kits aim to simplify the performance of microbial viability determination but often still need adaptation to the specific organism of interest and/or the instruments available to the researcher. This review explores the meaning of microbial viability and offers guidance in the selection and interpretation of viability testing methods.

Identification and characterization of high-flux-control genes of yeast through competition analyses in continuous cultures

Using competition experiments in continuous cultures grown in different nutrient environments (glucose limited, ammonium limited, phosphate limited and white grape juice), we identified genes that show haploinsufficiency phenotypes (reduced growth rate when hemizygous) or haploproficiency phenotypes (increased growth rate when hemizygous). Haploproficient genes (815, 1,194, 733 and 654 in glucose-limited, ammonium-limited, phosphate-limited and white grape juice environments, respectively) frequently show that phenotype in a specific environmental context. For instance, genes encoding components of the ubiquitination pathway or the proteasome show haploproficiency in nitrogen-limited conditions where protein conservation may be beneficial. Haploinsufficiency is more likely to be observed in all environments, as is the case with genes determining polar growth of the cell. Haploproficient genes seem randomly distributed in the genome, whereas haploinsufficient genes (685, 765, 1,277 and 217 in glucose-limited, ammonium-limited, phosphate-limited and white grape juice environments, respectively) are over-represented on chromosome III. This chromosome determines a yeasts mating type, and the concentration of haploinsufficient genes there may be a mechanism to prevent its loss.

Discrimination of Modes of Action of Antifungal Substances by Use of Metabolic Footprinting

ABSTRACT Diploid cells of Saccharomyces cerevisiae were grown under controlled conditions with a Bioscreen instrument, which permitted the essentially continuous registration of their growth via optical density measurements. Some cultures were exposed to concentrations of a number of antifungal substances with different targets or modes of action (sterol biosynthesis, respiratory chain, amino acid synthesis, and the uncoupler). Culture supernatants were taken and analyzed for their “metabolic footprints” by using direct-injection mass spectrometry. Discriminant function analysis and hierarchical cluster analysis allowed these antifungal compounds to be distinguished and classified according to their modes of action. Genetic programming, a rule-evolving machine learning strategy, allowed respiratory inhibitors to be discriminated from others by using just two masses. Metabolic footprinting thus represents a rapid, convenient, and information-rich method for classifying the modes of action of antifungal substances.

Variable selection and multivariate methods for the identification of microorganisms by flow cytometry.

BACKGROUND When exploited fully, flow cytometry can be used to provide multiparametric data for each cell in the sample of interest. While this makes flow cytometry a powerful technique for discriminating between different cell types, the data can be difficult to interpret. Traditionally, dual-parameter plots are used to visualize flow cytometric data, and for a data set consisting of seven parameters, one should examine 21 of these plots. A more efficient method is to reduce the dimensionality of the data (e.g., using unsupervised methods such as principal components analysis) so that fewer graphs need to be examined, or to use supervised multivariate data analysis methods to give a prediction of the identity of the analyzed particles. MATERIALS AND METHODS We collected multiparametric data sets for microbiological samples stained with six cocktails of fluorescent stains. Multivariate data analysis methods were explored as a means of microbial detection and identification. RESULTS We show that while all cocktails and all methods gave good accuracy of predictions (>94%), careful selection of both the stains and the analysis method could improve this figure (to > 99% accuracy), even in a data set that was not used in the formation of the supervised multivariate calibration model. CONCLUSIONS Flow cytometry provides a rapid method of obtaining multiparametric data for distinguishing between microorganisms. Multivariate data analysis methods have an important role to play in extracting the information from the data obtained. Artificial neural networks proved to be the most suitable method of data analysis.

Predatory activity of Myxococcus xanthus outer-membrane vesicles and properties of their hydrolase cargo

The deltaproteobacterium Myxococcus xanthus predates upon members of the soil microbial community by secreting digestive factors and lysing prey cells. Like other Gram-negative bacteria, M. xanthus produces outer membrane vesicles (OMVs), and we show here that M. xanthus OMVs are able to kill Escherichia coli cells. The OMVs of M. xanthus were found to contain active proteases, phosphatases, other hydrolases and secondary metabolites. Alkaline phosphatase activity was found to be almost exclusively associated with OMVs, implying that there is active targeting of phosphatases into OMVs, while other OMV components appear to be packaged passively. The kinetic properties of OMV alkaline phosphatase suggest that there may have been evolutionary adaptation of OMV enzymes to a relatively indiscriminate mode of action, consistent with a role in predation. In addition, the observed regulation of production, and fragility of OMV activity, may protect OMV-producing cells from exploitation by M. xanthus cheating genotypes and/or other competitors. Killing of E. coli by M. xanthus OMVs was enhanced by the addition of a fusogenic enzyme (glyceraldehyde-3-phosphate dehydrogenase; GAPDH), which triggers fusion of vesicles with target membranes within eukaryotic cells. This suggests that the mechanism of prey killing involves OMV fusion with the E. coli outer membrane. M. xanthus secretes GAPDH, which could potentially modulate the fusion of co-secreted OMVs with prey organisms in nature, enhancing their predatory activity.

Introduction to the dielectric estimation of cellular biomass in real time, with special emphasis on measurements at high volume fractions

The equations that describe the magnitude of the β-dielectric dispersion of biological cell suspensions are introduced. It is then demonstrated how this magnitude can be used to monitor cellular biomass concentrations in real time. These equations are then shown accurately to describe experimental data obtained over a wide range of cell sizes and volume fractions.

Estimation of Microbial Viability Using Flow Cytometry

For microorganisms in particular, viability is a term that is difficult to define and a state consequently difficult to measure. The traditional (and gold‐standard) usage equates viability and culturability (i.e., the ability to multiply), but the process of determining culturability is often too slow. Flow cytometry provides the opportunity to make rapid and quantitative measurements of dye uptake in large numbers of cells, and we can therefore exploit the flow cytometric approach to evaluate so‐called viability stains and to develop protocols for more routine assessments of microbial viability. This unit is primarily commentary, but several basic protocols have been included to ensure that users have a firm basis for attempting these reasonably difficult assays on traditional flow cytometer instruments. What is clear is that each assay must be carefully validated with the particular microorganism of interest before being applied in any research, clinical, or service form.

Recent advances in the analysis of individual microbial cells.

Muller, S., Davey, H. (2009). Editorial: Recent advances in the analysis of individual microbial cells. Cytometry Part A, 75A, 83-85. Editorial IMPF: 03.03 RONO: 00

Fluorescent brighteners: Novel stains for the flow cytometric analysis of microorganisms

Flow cytometry is a rapid method for measuring the optical properties of individual cells. The technique has found great utility in the study of mammalian cells, but microbiological applications have been more limited. We here show that UV-excited fluorescent whitening agents, in particular Tinopal CBS-X, are effective stains for both vegetative microbial cells and for spores of Gram-positive bacteria. Pretreatment of samples with ethanol speeds the staining process. Under favourable conditions, Tinopal CBS-X may be used to discriminate among organisms, a fact that may be useful when screening for a target microorganism against a high biological background.

Surviving the heat: heterogeneity of response in Saccharomyces cerevisiae provides insight into thermal damage to the membrane

Environmental heat stress impacts on the physiology and viability of microbial cells with concomitant implications for microbial activity and diversity. Previously, it has been demonstrated that gradual heating of Saccharomyces cerevisiae induces a degree of thermal resistance, whereas a heat shock results in a high level of cell death. Here, we show that the impact of exogenous nutrients on acquisition of thermal resistance differs between strains. Using single-cell methods, we demonstrate the extent of heterogeneity of the heat-stress response within populations of yeast cells and the presence of subpopulations that are reversibly damaged by heat stress. Such cells represent potential for recovery of entire populations once stresses are removed. The results show that plasma membrane permeability and potential are key factors involved in cell survival, but thermal resistance is not related to homeoviscous adaptation of the plasma membrane. These results have implications for growth and regrowth of populations experiencing environmental heat stress and our understanding of impacts at the level of the single cell. Given the important role of microbes in biofuel production and bioremediation, a thorough understanding of the impact of stress responses of populations and individuals is highly desirable.