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

Abstract

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.

Rose bengal uptake by E. faecalis and F. nucleatum and light-mediated antibacterial activity measured by flow cytometry

“…Although this technique is still considered as the gold-standard for bacterial viability assessment, it fails to identify subpopulations of viable but not cultivable (VBNC) bacteria exposed to antimicrobial agents [21,22]. Unlike culture plating, flow cytometry (FCM) in combination with fluorescent labeling of cells can distinguish different physiological states of single-cell microorganisms [23,24].…”

Viability states of bacteria—Specific mechanisms of selected probes

“…Indicators for cell viability can be activities of enzymes involved in substrate uptake and cleavage, the energy status of a cell and the integrity states of cell wall and cell membranes. These individual characteristics can be followed both with microscopic techniques and flow cytometry by using fluorescent or fluorogenic dyes, which characteristically deliver information on specific cell states (1). Besides basic knowledge on dye action the researchers have to keep in mind the tremendous diversity of bacteria with regard to structure and function.…”

Individual-based modeling of soil organic matter in NetLogo: Transparent, user-friendly, and open

“…Future studies may incorporate also micro scale observations , which have become possible through a variety of novel analytical techniques (e.g. BrehmStecher and Johnson, 2004;Müller and Nebe-von-Caron, 2010;Watrous and Dorrestein, 2011). Such observational data can be beneficial as direct input (providing parameter values or initial conditions), but also for inverse modeling (being compared to emergent model output to derive parameter values or process definitions).…”