Work in the group of JWV is supported by an EU Marie-Curie Reintegration Fellowship, a Sysmo2 Grant (NWO-ALW/ERASysBio), a Horizon grant (ZonMW) and by a VENI fellowship (NWO-ALW). The group of OPK is supported by several STW grants (NWO), a SYSMO1 (IGdeJ) and SYSMO2 grant, an ESF Eurocores SynBio grant (SynMod) and by the Kluyver Center for Genomics of Industrial Fermentation and the Top Institute for Food and Nutrition.

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No conflicts of interest declared.

In contrast to many other single cell techniques, the time-lapse fluorescence microscopy method described here can be used to follow the history of a specific cell with regard to its ancestors, its behaviour, and division events. In combination with fluorescently labelled target promoters or proteins, specific developmental pathway activation can be followed in time and protein localization as well as protein dynamics can be monitored during bacterial development.
As indicated above, studies concentrating on different bacterial species can be performed by adapting the growth conditions according to the requirements for a specific bacterium. The only limitations we encountered are related to the growth conditions and sample size. Due to a sealed environment, medium conditions cannot be changed during the experiment. Furthermore, a maximum of four strains per experiment can be monitored efficiently. 
Considering a few critical steps, the single cell analysis method described here can easily be applied using any automated microscope. In the following, an overview of these critical steps will be given. Detailed information can be found in the main text. General preparation: It is wise to check the autofocus settings required for a specific bacterium prior to the experiment. Likewise, approximate optimal settings for the visualization of fluorescence should be determined in advance, if possible. Furthermore, following a prepared time-line helps to have all material ready to use in time (pre-warming the microscope chamber, programming the microscope settings, preparing the slide one hour before the cells are in the desired growth phase, see Figure 1). Growth of B. subtilis in TLM and CDM: TLM and CDM are chemically defined starvation media in which B. subtilis only grows slowly. The time-period in which the cells are grown in the media might have to be prolonged depending on the specific strain. The slow growth prevents cells from piling up on each other. Preparation of the microscope sample: Air bubbles between the gene frame, the glass slide and the cover slip have to be prevented to prevent extensive drying of the agarose-based medium. The same holds for the medium/cover slip interface. It is crucial to let the cells dry enough, to prevent swimming and/or multiple layer growth. Time-lapse fluorescence microscopy: Pre-warming of the slide as well as the environmental chamber is crucial to prevent major autofocus problems. Cells should be selected in the middle of an agar pad, since these have the highest chance to stay in field and focus during the experiment (provided the sample was dried well enough). A maximum of 10 locations per experiments still works properly. After having selected the first cell of interest only use the software to adjust the focus (see text for details). Check whether the cells are still in focus during the first hours of the experiment in 30 min intervals. Analysis: It is important to check prior to extended analysis procedures whether the background of the medium has similar values in the fluorescence channels. Small dust particles, medium components, dirty lenses or tiny agarose clumps can contribute to locally increased fluorescence, making the movie difficult or impossible to analyse. Trouble shooting: If the cells grow on top of each other, this might either indicate that the coverslip was attached too soon or that the medium is not suited for growth of microcolony monolayers. If cells of interest continuously die prematurely, whereas other cells on the slide divide happily, you might want to check whether you put the UV-filter in position. It might also help to decrease the exposure time or light intensity during long experiments.

<table border="1">
	<tbody>
		<tr>
			<th>Name of the reagent	</th>
			<th>Company	</th>
			<th>Catalogue number	</th>
			<th>Comments (optional)</th>
		</tr>
		
		<tr>
			<td>Gene Frame	</td>
			<td>ABgene	</td>
			<td>AB-0578	</td>
			<td>1.7 x 2.8 cm</td>
		</tr>
		
		<tr>
			<td>high-resolution low melting agarose	</td>
			<td>Sigma	</td>
			<td>A4718</td>
			<td>&nbsp;</td>
		</tr>
		
		<tr>
			<td>big cover slip	</td>
			<td>several		</td>
			<td>&nbsp;</td>
			<td>24 x 50 mm</td>
		</tr>
		
		<tr>
			<td>if desired, membrane dye, e.g. FM 5-95</td>
			<td>Invitrogen	</td>
			<td>T23360</td>
			<td>other membrane dyes are also available: <a href="http://probes.invitrogen.com/media/pis/mp34653.pdf">http://probes.invitrogen.com/media/pis/mp34653.pdf</a></td>
		</tr>
		
		<tr>
			<td>Time-lapse microscope with environmental chamber</td>
			<td>several</td>
			<td>&nbsp;</td>
			<td>see details for our device in the corresponding sections</td>
		</tr>
	</tbody>
</table>

live cell imaging of bacillus subtilis and streptococcus pneumoniae using automated time-lapse microscopy

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.

Watch this Scientific Journal Video about Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy at JoVE.com

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

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42.0K Views.  University of Groningen. This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.