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A multi-platform flow device for microbial (co-) cultivation and microscopic analysis.

Hesselman MC, Odoni DI, Ryback BM, de Groot S, van Heck RG, Keijsers J, Kolkman P, Nieuwenhuijse D, van Nuland YM, Sebus E, Spee R, de Vries H, Wapenaar MT, Ingham CJ, Schroën K, Martins dos Santos VA, Spaans SK, Hugenholtz F, van Passel MW - PLoS ONE (2012)

Bottom Line: The development of materials with specialized chemical and geometric properties has opened up new possibilities in the study of previously unculturable microorganisms and has facilitated the design of elegant, high-throughput experimental set-ups.Within the context of the international Genetically Engineered Machine (iGEM) competition, we set out to design, manufacture, and implement a flow device that can accommodate multiple growth platforms, that is, a silicon nitride based microsieve and a porous aluminium oxide based microdish.The device was designed to be affordable, reusable, and above all, versatile.

View Article: PubMed Central - PubMed

Affiliation: Wageningen UR iGEM 2011 Team, Wageningen University, Wageningen, The Netherlands.

ABSTRACT
Novel microbial cultivation platforms are of increasing interest to researchers in academia and industry. The development of materials with specialized chemical and geometric properties has opened up new possibilities in the study of previously unculturable microorganisms and has facilitated the design of elegant, high-throughput experimental set-ups. Within the context of the international Genetically Engineered Machine (iGEM) competition, we set out to design, manufacture, and implement a flow device that can accommodate multiple growth platforms, that is, a silicon nitride based microsieve and a porous aluminium oxide based microdish. It provides control over (co-)culturing conditions similar to a chemostat, while allowing organisms to be observed microscopically. The device was designed to be affordable, reusable, and above all, versatile. To test its functionality and general utility, we performed multiple experiments with Escherichia coli cells harboring synthetic gene circuits and were able to quantitatively study emerging expression dynamics in real-time via fluorescence microscopy. Furthermore, we demonstrated that the device provides a unique environment for the cultivation of nematodes, suggesting that the device could also prove useful in microscopy studies of multicellular microorganisms.

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Schematic representation of the flow device.A) Schematic representation of the flow device, with the dimensions in mm. Depicted in red and blue are the in- and outflow channels of the top compartment (light green). The respective in- and outflow channels of the lower compartment (yellow) are given in purple and dark green. B) Electron microscopy image of a microsieve. C) Electron microscopy image of a microdish. See File S1 for more views of the device.
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pone-0036982-g001: Schematic representation of the flow device.A) Schematic representation of the flow device, with the dimensions in mm. Depicted in red and blue are the in- and outflow channels of the top compartment (light green). The respective in- and outflow channels of the lower compartment (yellow) are given in purple and dark green. B) Electron microscopy image of a microsieve. C) Electron microscopy image of a microdish. See File S1 for more views of the device.

Mentions: The device is made from polymethylmethacrylate (perspex), a transparent, durable, autoclavable, and inexpensive material (Figure 1). The in- and outlets are threaded, which allows plugs and tubing to be screwed in securely. The material is resistant to ethanol and chlorine-based cleaning solutions, and can be cleaned with these to sterilize the device between experiments (http://www.gehr.de/dyndata/PMMA_engl.pdf). The device is not as susceptible to biofouling as microfluidic chips due to the internal dimensions that allow cleaning solutions to remove any biological residue. We did not observe any residual contamination after repeated experiments (data not shown). The distance between the socket and the top of the chamber is ∼1 mm. This proximity allows for a magnification with a 20x objective for a total magnification of 200x using an Olympus Microscope BX41, which allows the detection of fluorescence emitted by small agglomerations of cells. Detection of single bacterial cells may be also possible using objectives with higher magnification as long as the minimum focal depth is no less than 1 mm, such as 40x and 60x water-immersion objectives of the Olympus LUMPLFLN-W series. The dimensions of the chamber were chosen such that both growing platforms could be accommodated. The resulting total volume of the main chamber is ∼0.5 ml. The device can be sealed with a thin glass slide using Bison mastic silicon kit, which is removable and enables the reuse of the device after an experiment. The flow device was used with a ProSense syringe pump NE1000X2 for the flow of media or buffers which is connected to the device via transparent silicon tubing. The flow device was designed using Google SketchUp (http://sketchup.google.com), and manufactured with a standard workbench computer controlled drill in the fine mechanical workshop of Wageningen University. The sketch files of the device are available for users to customize and manufacture their own flow device (File S1). The cost of a single, reusable device, excluding a microsieve or microdish, can be approximated to be <150 Euros, the bulk of which can be attributed to manual labor costs.


A multi-platform flow device for microbial (co-) cultivation and microscopic analysis.

Hesselman MC, Odoni DI, Ryback BM, de Groot S, van Heck RG, Keijsers J, Kolkman P, Nieuwenhuijse D, van Nuland YM, Sebus E, Spee R, de Vries H, Wapenaar MT, Ingham CJ, Schroën K, Martins dos Santos VA, Spaans SK, Hugenholtz F, van Passel MW - PLoS ONE (2012)

Schematic representation of the flow device.A) Schematic representation of the flow device, with the dimensions in mm. Depicted in red and blue are the in- and outflow channels of the top compartment (light green). The respective in- and outflow channels of the lower compartment (yellow) are given in purple and dark green. B) Electron microscopy image of a microsieve. C) Electron microscopy image of a microdish. See File S1 for more views of the device.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3351485&req=5

pone-0036982-g001: Schematic representation of the flow device.A) Schematic representation of the flow device, with the dimensions in mm. Depicted in red and blue are the in- and outflow channels of the top compartment (light green). The respective in- and outflow channels of the lower compartment (yellow) are given in purple and dark green. B) Electron microscopy image of a microsieve. C) Electron microscopy image of a microdish. See File S1 for more views of the device.
Mentions: The device is made from polymethylmethacrylate (perspex), a transparent, durable, autoclavable, and inexpensive material (Figure 1). The in- and outlets are threaded, which allows plugs and tubing to be screwed in securely. The material is resistant to ethanol and chlorine-based cleaning solutions, and can be cleaned with these to sterilize the device between experiments (http://www.gehr.de/dyndata/PMMA_engl.pdf). The device is not as susceptible to biofouling as microfluidic chips due to the internal dimensions that allow cleaning solutions to remove any biological residue. We did not observe any residual contamination after repeated experiments (data not shown). The distance between the socket and the top of the chamber is ∼1 mm. This proximity allows for a magnification with a 20x objective for a total magnification of 200x using an Olympus Microscope BX41, which allows the detection of fluorescence emitted by small agglomerations of cells. Detection of single bacterial cells may be also possible using objectives with higher magnification as long as the minimum focal depth is no less than 1 mm, such as 40x and 60x water-immersion objectives of the Olympus LUMPLFLN-W series. The dimensions of the chamber were chosen such that both growing platforms could be accommodated. The resulting total volume of the main chamber is ∼0.5 ml. The device can be sealed with a thin glass slide using Bison mastic silicon kit, which is removable and enables the reuse of the device after an experiment. The flow device was used with a ProSense syringe pump NE1000X2 for the flow of media or buffers which is connected to the device via transparent silicon tubing. The flow device was designed using Google SketchUp (http://sketchup.google.com), and manufactured with a standard workbench computer controlled drill in the fine mechanical workshop of Wageningen University. The sketch files of the device are available for users to customize and manufacture their own flow device (File S1). The cost of a single, reusable device, excluding a microsieve or microdish, can be approximated to be <150 Euros, the bulk of which can be attributed to manual labor costs.

Bottom Line: The development of materials with specialized chemical and geometric properties has opened up new possibilities in the study of previously unculturable microorganisms and has facilitated the design of elegant, high-throughput experimental set-ups.Within the context of the international Genetically Engineered Machine (iGEM) competition, we set out to design, manufacture, and implement a flow device that can accommodate multiple growth platforms, that is, a silicon nitride based microsieve and a porous aluminium oxide based microdish.The device was designed to be affordable, reusable, and above all, versatile.

View Article: PubMed Central - PubMed

Affiliation: Wageningen UR iGEM 2011 Team, Wageningen University, Wageningen, The Netherlands.

ABSTRACT
Novel microbial cultivation platforms are of increasing interest to researchers in academia and industry. The development of materials with specialized chemical and geometric properties has opened up new possibilities in the study of previously unculturable microorganisms and has facilitated the design of elegant, high-throughput experimental set-ups. Within the context of the international Genetically Engineered Machine (iGEM) competition, we set out to design, manufacture, and implement a flow device that can accommodate multiple growth platforms, that is, a silicon nitride based microsieve and a porous aluminium oxide based microdish. It provides control over (co-)culturing conditions similar to a chemostat, while allowing organisms to be observed microscopically. The device was designed to be affordable, reusable, and above all, versatile. To test its functionality and general utility, we performed multiple experiments with Escherichia coli cells harboring synthetic gene circuits and were able to quantitatively study emerging expression dynamics in real-time via fluorescence microscopy. Furthermore, we demonstrated that the device provides a unique environment for the cultivation of nematodes, suggesting that the device could also prove useful in microscopy studies of multicellular microorganisms.

Show MeSH
Related in: MedlinePlus