Susann Müller

Functional single-cell analyses: flow cytometry and cell sorting of microbial populations and communities.

The still poorly explored world of microbial functioning is about to be uncovered by a combined application of old and new technologies. Bacteria, especially, are still in the dark with respect to their phylogenetic affiliations as well as their metabolic capabilities and functions. However, with the advent of sophisticated flow cytometric and cell sorting technologies in microbiological labs, there is now the possibility to gain this knowledge at the single-cell level without cumbersome cultivation approaches. Cytometry also facilitates the understanding of physiological diversity in seemingly likewise acting populations. Both individuality and diversity lead to the complex and concerted actions of microbial consortia. This review provides an overview of the state of the art in the field. It deals with the handling of microorganisms from the very beginning (i.e. sampling, and detachment and fixation procedures) and goes on to discuss the pitfalls and problems in analysing cells without any further treatment. If information cannot be gained by specific staining procedures, phylogenetic technologies, transcriptomic and proteomic approaches may be options for achieving advanced insights. All in all, flow cytometry will be a mediator technology to gain a deeper insight into the heterogeneity of populations and the functioning of microbial communities.

Limits of propidium iodide as a cell viability indicator for environmental bacteria.

Viability measurements of individual bacteria are applied in various scopes of research and industry using approaches where propidium iodide (PI) serves as dead cell indicator. The reliability of PI uptake as a cell viability indicator for dead (PI permeable) and viable (PI impermeable) bacteria was tested using two soil bacteria, the gram−Sphingomonas sp. LB126 and the gram+Mycobacterium frederiksbergense LB501T.

Population Dynamics within a Microbial Consortium during Growth on Diesel Fuel in Saline Environments

ABSTRACT The diversity and dynamics of a bacterial community extracted from an exploited oil field with high natural soil salinity near Comodoro Rivadavia in Patagonia (Argentina) were investigated. Community shifts during long-term incubation with diesel fuel at four salinities between 0 and 20% NaCl were monitored by single-strand conformation polymorphism community fingerprinting of the PCR-amplified V4-V5 region of the 16S rRNA genes. Information obtained by this qualitative approach was extended by flow cytometric analysis to follow quantitatively the dynamics of community structures at different salinities. Dominant and newly developing clusters of individuals visualized via their DNA patterns versus cell sizes were used to identify the subcommunities primarily involved in the degradation process. To determine the most active species, subcommunities were separated physically by high-resolution cell sorting and subsequent phylogenetic identification by 16S rRNA gene sequencing. Reduced salinity favored the dominance of Sphingomonas spp., whereas at elevated salinities, Ralstonia spp. and a number of halophilic genera, including Halomonas, Dietzia, and Alcanivorax, were identified. The combination of cytometric sorting with molecular characterization allowed us to monitor community adaptation and to identify active and proliferating subcommunities.

Electroactive mixed culture derived biofilms in microbial bioelectrochemical systems: the role of pH on biofilm formation, performance and composition.

The pH-value played a crucial role for the development and current production of anodic microbial electroactive biofilms. It was demonstrated that only a narrow pH-window, ranging from pH 6 to 9, was suitable for growth and operation of biofilms derived from pH-neutral wastewater. Any stronger deviation from pH neutral conditions led to a substantial decrease in the biofilm performance. Thus, average current densities of 151, 821 and 730 μA cm(-2) were measured for anode biofilms grown and operated at pH 6, 7 and 9 respectively. The microbial diversity of the anode chamber community during the biofilm selection process was studied using the low cost method flow-cytometry. Thereby, it was demonstrated that the pH value as well as the microbial inocula had an impact on the resulting anode community structure. As shown by cyclic voltammetry the electron transfer thermodynamics of the biofilms was strongly depending on the solutions pH-value.

Origin and analysis of microbial population heterogeneity in bioprocesses.

Heterogeneity of industrial production cultures is accepted to a certain degree; however, the underlying mechanisms are seldom perceived or included in the development of new bioprocess control strategies. Population heterogeneity and its basics, perceptible in the diverse proficiency of cells, begins with asymmetric birth and is found to recess during the life cycle. Since inefficient subpopulations have significant impact on the productivity of industrial cultures, cellular heterogeneity needs to be detected and quantified by using high speed detection tools like flow cytometry. Possible origins of population heterogeneity, sophisticated fluorescent techniques for detection of individual cell states, and cutting-edge Omics-technologies for extended information beyond the resolution of fluorescent labelling are highlighted.

From multi-omics to basic structures of biological systems.

Susann Müller is a professor in Microbiology and works at the Helmholtz Centre for Environmental Research – UFZ in Leipzig, Germany. She has more than 20 years of experience in microbial single cell analytics starting with applications mainly rooted in biotechnology to research performed on complex natural communities. Methods to combine microbial cytomics with other omics technologies were established in the recent years. She was president of the German Society for Cytometry (DGfZ, 2008–2010) and is Associate Editor for Microbiology for Cytometry Part A.

Viability states of bacteria - specific mechanisms of selected probes.

Single cell techniques like flow cytometry combined with viability staining can help to obtain information on viability states of bacteria. Many fluorescent dyes are available for this purpose and can be chosen according to the available excitation source, the species used, and the background of scientific questions and relevant specifications. Within this short overview, we focus on two diverse groups of bacteria: the gram− Escherichia coli and representatives of the gram+ Mycobacterium to demonstrate differences and similarities in dye uptake principles, processing and binding. We call for attention to possible diverse responses of different species to various viability assays. The cell surface structure of bacteria and the chemical properties of fluorescent probes considerably determine the success of a certain staining practice. Particular focus was drawn on analysis of membrane integrity, uptake of substrates and transformation of fluorogenic substrates.

Dynamics of Polyphosphate-Accumulating Bacteria in Wastewater Treatment Plant Microbial Communities Detected via DAPI (4′,6′-Diamidino-2-Phenylindole) and Tetracycline Labeling

ABSTRACT Wastewater treatment plants with enhanced biological phosphorus removal represent a state-of-the-art technology. Nevertheless, the process of phosphate removal is prone to occasional failure. One reason is the lack of knowledge about the structure and function of the bacterial communities involved. Most of the bacteria are still not cultivable, and their functions during the wastewater treatment process are therefore unknown or subject of speculation. Here, flow cytometry was used to identify bacteria capable of polyphosphate accumulation within highly diverse communities. A novel fluorescent staining technique for the quantitative detection of polyphosphate granules on the cellular level was developed. It uses the bright green fluorescence of the antibiotic tetracycline when it complexes the divalent cations acting as a countercharge in polyphosphate granules. The dynamics of cellular DNA contents and cell sizes as growth indicators were determined in parallel to detect the most active polyphosphate-accumulating individuals/subcommunities and to determine their phylogenetic affiliation upon cell sorting. Phylotypes known as polyphosphate-accumulating organisms, such as a “Candidatus Accumulibacter”-like phylotype, were found, as well as members of the genera Pseudomonas and Tetrasphaera. The new method allows fast and convenient monitoring of the growth and polyphosphate accumulation dynamics of not-yet-cultivated bacteria in wastewater bacterial communities.

Cytometric fingerprinting for analyzing microbial intracommunity structure variation and identifying subcommunity function

Functions of complex natural microbial communities are realized by single cells that contribute differently to the overall performance of a community. Usually, molecular and, more recently, deep-sequencing techniques are used for detailed but resource-consuming phylogenetic or functional analyses of microbial communities. Here we present a method for analyzing dynamic community structures that rapidly detects functional (rather than phylogenetic) coherent subcommunities by monitoring changes in cell-specific and abiotic microenvironmental parameters. The protocol involves the use of flow cytometry to analyze elastic light scattering and fluorescent cell labeling, with subsequent determination of cell gate abundance and finally the creation of a cytometric community fingerprint. Abiotic parameter analysis data are correlated with the dynamic cytometric fingerprint to obtain a time-bound functional heat map. The map facilitates the identification of activity hot spots in communities, which can be further resolved by subsequent cell sorting of key subcommunities and concurrent phylogenetic analysis (terminal restriction fragment length polymorphism, tRFLP). The cytometric fingerprint information is based on gate template settings and the functional heat maps are created using an R script. Cytometric fingerprinting and evaluation can be accomplished in 1 d, and additional subcommunity composition information can be obtained in a further 6 d.

Modes of cytometric bacterial DNA pattern: a tool for pursuing growth

Abstract.  Analyses of DNA pattern provide an excellent tool to determine activity states of bacteria. Bacterial cell cycle behaviour is generally different from the eukaryotic one and is pre‐determined by the bacterias diversity within the phylogenetic tree, and their metabolic traits. As a result, every species creates its specific proliferation pattern that differs from every other one. Up to now, just few bacterial species have been investigated and little information is available concerning DNA cycling even in already known species. This prevents understanding of the complexity and diversity of ongoing bacterial interactions in many ecosystems or in biotechnology. Flow cytometry is the only possible technique to shed light on the dynamics of bacterial communities and DNA patterns will help to unlock the hidden principles of their life. This review provides basic knowledge about the molecular background of bacterial cell cycling, discusses modes of cell cycle phases and presents techniques to both obtain DNA patterns and to combine the contained information with physiological cell states.