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A microscope automated fluidic system to study bacterial processes in real time.

Ducret A, Maisonneuve E, Notareschi P, Grossi A, Mignot T, Dukan S - PLoS ONE (2009)

Bottom Line: In fact, in many cases agar is the experimental solid substratum on which bacteria can move or even grow.By studying several examples, we show that this system allows real time analysis of a broad array of biological processes such as growth, development and motility.Thus, the flow chamber system will be an essential tool to study any process that take place on an agar surface at the single cell level.

View Article: PubMed Central - PubMed

Affiliation: Aix Marseille Université, Laboratoire de Chimie Bactérienne (UPR 9043), Institut de Microbiologie de la Méditerranée (IFR 88), CNRS, 31, Chemin Joseph Aiguier, Marseille, France.

ABSTRACT
Most time lapse microscopy experiments studying bacterial processes ie growth, progression through the cell cycle and motility have been performed on thin nutrient agar pads. An important limitation of this approach is that dynamic perturbations of the experimental conditions cannot be easily performed. In eukaryotic cell biology, fluidic approaches have been largely used to study the impact of rapid environmental perturbations on live cells and in real time. However, all these approaches are not easily applicable to bacterial cells because the substrata are in all cases specific and also because microfluidics nanotechnology requires a complex lithography for the study of micrometer sized bacterial cells. In fact, in many cases agar is the experimental solid substratum on which bacteria can move or even grow. For these reasons, we designed a novel hybrid micro fluidic device that combines a thin agar pad and a custom flow chamber. By studying several examples, we show that this system allows real time analysis of a broad array of biological processes such as growth, development and motility. Thus, the flow chamber system will be an essential tool to study any process that take place on an agar surface at the single cell level.

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Related in: MedlinePlus

SodA-mcherry induction in cells containing inclusion bodies during MV injection.A) Measurement of SodA induction after Methyl Viologen injection and removal (indicated as grey shade) in cells carrying the SodA-mcherry fusion. Each colored curve represents the kinetics of expression of a given single cell B) Measurement of SodA induction in cells that carry polar foci of the protein aggregation marker IbpA-YFP. C) Sequence of images showing several rounds of cell division from a single mother cell carrying a polar IbpA-YFP polar focus; Top: phase images. Bottom: overlay of YFP (magenta) and mCherry fluorescent signals. Cell contours retrieved by the annotation software are added for clarity (yellow cells containing IbpA-YFP clusters and green cells not containing the clusters).
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pone-0007282-g003: SodA-mcherry induction in cells containing inclusion bodies during MV injection.A) Measurement of SodA induction after Methyl Viologen injection and removal (indicated as grey shade) in cells carrying the SodA-mcherry fusion. Each colored curve represents the kinetics of expression of a given single cell B) Measurement of SodA induction in cells that carry polar foci of the protein aggregation marker IbpA-YFP. C) Sequence of images showing several rounds of cell division from a single mother cell carrying a polar IbpA-YFP polar focus; Top: phase images. Bottom: overlay of YFP (magenta) and mCherry fluorescent signals. Cell contours retrieved by the annotation software are added for clarity (yellow cells containing IbpA-YFP clusters and green cells not containing the clusters).

Mentions: Inside the chamber, E. coli cells grew normally with doubling times comparable to those measured in liquid assays (Fig. 3C, Movie S1).At the beginning of the experiment about 50% of the cells contained at least one IbpA-YFP fluorescent foci at cellular poles, as previously described [12]. The IbpA-YFP foci rarely moved from their initial position so that, upon cell division, the cluster was only inherited by one of the daughter cells [12]. Interestingly, IbpA-YFP clusters were not newly formed in the course of the experiment (Fig. 3C, Movie S1). Injection of MV, led to very synchronous induction of SodA in all cells after a lag time of 7 min (Fig. 3A). To obtain reliable and quantitative measurements of SodA induction in cells with or without protein aggregates, we developed a script allowing automated and selective analysis of cells containing IbpA-YFP foci or not (see Methods). In cells containing IbpA-YFP the response to MV was indistinguishable from cells that contained no aggregates.


A microscope automated fluidic system to study bacterial processes in real time.

Ducret A, Maisonneuve E, Notareschi P, Grossi A, Mignot T, Dukan S - PLoS ONE (2009)

SodA-mcherry induction in cells containing inclusion bodies during MV injection.A) Measurement of SodA induction after Methyl Viologen injection and removal (indicated as grey shade) in cells carrying the SodA-mcherry fusion. Each colored curve represents the kinetics of expression of a given single cell B) Measurement of SodA induction in cells that carry polar foci of the protein aggregation marker IbpA-YFP. C) Sequence of images showing several rounds of cell division from a single mother cell carrying a polar IbpA-YFP polar focus; Top: phase images. Bottom: overlay of YFP (magenta) and mCherry fluorescent signals. Cell contours retrieved by the annotation software are added for clarity (yellow cells containing IbpA-YFP clusters and green cells not containing the clusters).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2748647&req=5

pone-0007282-g003: SodA-mcherry induction in cells containing inclusion bodies during MV injection.A) Measurement of SodA induction after Methyl Viologen injection and removal (indicated as grey shade) in cells carrying the SodA-mcherry fusion. Each colored curve represents the kinetics of expression of a given single cell B) Measurement of SodA induction in cells that carry polar foci of the protein aggregation marker IbpA-YFP. C) Sequence of images showing several rounds of cell division from a single mother cell carrying a polar IbpA-YFP polar focus; Top: phase images. Bottom: overlay of YFP (magenta) and mCherry fluorescent signals. Cell contours retrieved by the annotation software are added for clarity (yellow cells containing IbpA-YFP clusters and green cells not containing the clusters).
Mentions: Inside the chamber, E. coli cells grew normally with doubling times comparable to those measured in liquid assays (Fig. 3C, Movie S1).At the beginning of the experiment about 50% of the cells contained at least one IbpA-YFP fluorescent foci at cellular poles, as previously described [12]. The IbpA-YFP foci rarely moved from their initial position so that, upon cell division, the cluster was only inherited by one of the daughter cells [12]. Interestingly, IbpA-YFP clusters were not newly formed in the course of the experiment (Fig. 3C, Movie S1). Injection of MV, led to very synchronous induction of SodA in all cells after a lag time of 7 min (Fig. 3A). To obtain reliable and quantitative measurements of SodA induction in cells with or without protein aggregates, we developed a script allowing automated and selective analysis of cells containing IbpA-YFP foci or not (see Methods). In cells containing IbpA-YFP the response to MV was indistinguishable from cells that contained no aggregates.

Bottom Line: In fact, in many cases agar is the experimental solid substratum on which bacteria can move or even grow.By studying several examples, we show that this system allows real time analysis of a broad array of biological processes such as growth, development and motility.Thus, the flow chamber system will be an essential tool to study any process that take place on an agar surface at the single cell level.

View Article: PubMed Central - PubMed

Affiliation: Aix Marseille Université, Laboratoire de Chimie Bactérienne (UPR 9043), Institut de Microbiologie de la Méditerranée (IFR 88), CNRS, 31, Chemin Joseph Aiguier, Marseille, France.

ABSTRACT
Most time lapse microscopy experiments studying bacterial processes ie growth, progression through the cell cycle and motility have been performed on thin nutrient agar pads. An important limitation of this approach is that dynamic perturbations of the experimental conditions cannot be easily performed. In eukaryotic cell biology, fluidic approaches have been largely used to study the impact of rapid environmental perturbations on live cells and in real time. However, all these approaches are not easily applicable to bacterial cells because the substrata are in all cases specific and also because microfluidics nanotechnology requires a complex lithography for the study of micrometer sized bacterial cells. In fact, in many cases agar is the experimental solid substratum on which bacteria can move or even grow. For these reasons, we designed a novel hybrid micro fluidic device that combines a thin agar pad and a custom flow chamber. By studying several examples, we show that this system allows real time analysis of a broad array of biological processes such as growth, development and motility. Thus, the flow chamber system will be an essential tool to study any process that take place on an agar surface at the single cell level.

Show MeSH
Related in: MedlinePlus