Torage [3]. Bacteria and fungi are able to form multicellular threedimensional communities

Torage [3]. Bacteria and fungi are able to form multicellular Rosiglitazone threedimensional communities, such as stalks [4,5], mats/biofilms?2014 Faria-Oliveira et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Faria-Oliveira et al. BMC Microbiology 2014, 14:244 http://www.biomedcentral.com/1471-2180/14/Page 2 of[6,7] and colonies [8-10]. These are supported and protected by an extracellular polymeric substance, or ECM, which originates from cellular synthesis and secretion processes, as well as from the lysis of embedded cells. When these microbial multicellular communities developed in natural environments, their ECM components account as well with contributions from the surrounding environment, for example from an infection host [11-13]. The ECM from Saccharomyces cerevisiae colonies revealed the presence of proteins [14], some of which highly glycosylated [15], which remain unidentified. In Candida ECM biofilms, several studies also reported the presence of proteins, as well as polysaccharides and DNA [16-18]. Importantly, an exopolysaccharide composed of -D-glucose and -D-glucose, -D-mannose, -L-rhamnose and N-acetyl glucosamine was identified in the ECM from a C. albicans biofilm recovered from an infected intrauterine device [12]. Additionally, the ECM-like substance from flocs of S. cerevisise, overexpressing FLO1, were shown to be composed of glucose and branched mannose [19]. Overall, the major players of the yeast colonies and biofilms remain unknown. Therefore, the identification of the molecules composing yeast ECM will contribute for the future understanding of cell-cell communication and other multicellular aggregation processes, namely quorum sensing. This work provides the foundations for the detailed identification of yeast ECM (yECM) components, presenting the development of a protocol to produce yECM, which robustness was challenged through inter-species reproducibility. Subsequently, methodologies for yECM extraction and fractionation were optimized, providing unprecedented analytical-grade protein and sugar fractions for chromatographic assessment. These methodological advances will open the way to future research on the processes and players of eukaryotic multicellular life-style.overcome this obstacle [7,20]. We took this concept one step further and inoculated enough cells to produce a whole-plate three-dimensional overlay. The method was tested with wild type strains of S. cerevisiae and C. albicans. Cells collected from a single colony were grown overnight until exponential growth phase, and these fully active cells were used to inoculate homogeneously the whole surface of the YPDa plates. The plates were firstly dried under a sterile air flow until the inoculum was properly absorbed and then incubated at 30 . The cell growth and overlay development was followed until the so-called «mature overlay» presented a high quantity of biomass. This happened as a result of 7 days of growth for the case of S. PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/7500280 cerevisiae, and 5 days growth in the case of C. albicans, consistently with the latter be.