Live Cell Imaging of <em>Bacillus subtilis</em> and <em>Streptococcus pneumoniae</em> using Automated Time-lapse Microscopy

Abstract

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3 ). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy). Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7 , which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Monitoring Spatial Segregation in Surface Colonizing Microbial Populations

“…The availability of a fluorescent toolbox for bacteria facilitates not only the study of heterogeneous gene expression 30,31 and protein localization 32 , but also the analysis of spatial distribution of strains within a population 8 . Fluorescent markers with sufficiently different excitation and emission wavelengths allow to distinctly localize two strains that otherwise are indistinguishable from each other when mixed.…”

Control of cell division in Streptococcus pneumoniae by the conserved Ser/Thr protein kinase StkP

“…Van-FL staining was performed as recently described (58). Time-lapse microscopy was basically performed as described (64). For more details, see SI Materials and Methods.…”

Ultrasensitivity of the Bacillus subtilis sporulation decision

“…S3F and S4E. Time-lapse microscopy was performed by the procedure of de Jong et al (63) using the fluorescence microscope with a temperature-controlled chamber from Solent Scientific. In brief, sporulating cells incubated for 1 h at 37°C in the medium of Sterlini and Mandelstam (61) were spotted on 1% (wt/vol) agarose pads containing the identical medium within a 25-μL Gene Frame and covered by a clean microscope slide coverslip.…”