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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

Layout of the microfluidic device.Cells are confined between the coverslip and a 0.5 mm thin layer of agar. The chamber is sealed with a transparent lid containing two entries allowing flow injections. Injected molecules reach the biological specimen by diffusion through the thin layer of agar.
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pone-0007282-g001: Layout of the microfluidic device.Cells are confined between the coverslip and a 0.5 mm thin layer of agar. The chamber is sealed with a transparent lid containing two entries allowing flow injections. Injected molecules reach the biological specimen by diffusion through the thin layer of agar.

Mentions: Most commercial uncoated chambers are not suitable for bacteria because the cells resuspend readily when flows are applied. Also, standard coatings are not suitable because the cells bind poorly or binding is toxic (such as poly-lysine substrates). In bacteria, most time-lapse experiments have been performed on agar, so we decided to develop a chamber that would use agar as the main substrate. On agar pads, bacterial cells such as E. coli cells bind non-specifically and divide with expected generation times over several hours [5], [12]. However, bacterial cells resuspend very rapidly when liquid is added atop the pad even in the absence of flow (data not shown). To overcome this problem we designed a chamber in which the cells are physically isolated from the liquid flow by the agar pad itself. Schematically, the cells are placed immediately on a coverslip and overlaid with a 0.5 mm thin layer of agar and left to dry gently to absorb the cells onto the agar substrate (Fig. 1). The chamber is then sealed with a transparent lid containing two entries allowing for flow injections (Fig. 1 & Fig. S1A). Flow injections are performed with home-developed computerized electronic injectors, allowing injection of flows ranging between 0,1–100 µL/s. Even at maximum flow rates, injections did not significantly detach the agar pad from the coverslip. Thus, in this chamber system, injected molecules reach the biological specimen by diffusion through the thin layer. In our system up to 12 different solutions can be injected sequentially in the course of the time-lapse experiment (Fig. S1B). The system is fully automated and driven by a custom software written in Visual Basic that runs under the Metamorph software (Fig. S1C) allowing both piloting of the system and image processing.


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)

Layout of the microfluidic device.Cells are confined between the coverslip and a 0.5 mm thin layer of agar. The chamber is sealed with a transparent lid containing two entries allowing flow injections. Injected molecules reach the biological specimen by diffusion through the thin layer of agar.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0007282-g001: Layout of the microfluidic device.Cells are confined between the coverslip and a 0.5 mm thin layer of agar. The chamber is sealed with a transparent lid containing two entries allowing flow injections. Injected molecules reach the biological specimen by diffusion through the thin layer of agar.
Mentions: Most commercial uncoated chambers are not suitable for bacteria because the cells resuspend readily when flows are applied. Also, standard coatings are not suitable because the cells bind poorly or binding is toxic (such as poly-lysine substrates). In bacteria, most time-lapse experiments have been performed on agar, so we decided to develop a chamber that would use agar as the main substrate. On agar pads, bacterial cells such as E. coli cells bind non-specifically and divide with expected generation times over several hours [5], [12]. However, bacterial cells resuspend very rapidly when liquid is added atop the pad even in the absence of flow (data not shown). To overcome this problem we designed a chamber in which the cells are physically isolated from the liquid flow by the agar pad itself. Schematically, the cells are placed immediately on a coverslip and overlaid with a 0.5 mm thin layer of agar and left to dry gently to absorb the cells onto the agar substrate (Fig. 1). The chamber is then sealed with a transparent lid containing two entries allowing for flow injections (Fig. 1 & Fig. S1A). Flow injections are performed with home-developed computerized electronic injectors, allowing injection of flows ranging between 0,1–100 µL/s. Even at maximum flow rates, injections did not significantly detach the agar pad from the coverslip. Thus, in this chamber system, injected molecules reach the biological specimen by diffusion through the thin layer. In our system up to 12 different solutions can be injected sequentially in the course of the time-lapse experiment (Fig. S1B). The system is fully automated and driven by a custom software written in Visual Basic that runs under the Metamorph software (Fig. S1C) allowing both piloting of the system and image processing.

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