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

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.

Figure 1. 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.

Figure 2. Cell retention on the microsieve.

Retained clusters of fluorescent E. coli cells on the sieve (100x magnification). The diagonal light grey bands are the permeable areas of the microsieve that contain the pores.

Figure 3. Oscillating GFP expression observed in microdish wells.

Fluorescent E. coli cells in the wells of the microdish showing variations in signal strength over time. The graph depicts variations plotted with the image analysis and processing tool ImageJ. The x-axis represents time, and the y-axis represents fluorescence (in arbitrary units and with a variance of maximally 0.01 for the normalised data of 5 wells). Below the graph are microscopic images of fluorescent bacteria in the cultivation chip wells at different intervals using identical illumination conditions and CCD camera exposure times. The time points at which the images were taken are indicated with an asterisk.

Figure 4. Co-cultivation of cells separated by a microsieve.

Increase of GFP expression of inducible cells on the sieve after inoculation of inducer cells below. Graph plotted with the image analysis and processing tool ImageJ. The x-axis corresponds to time and the y-axis shows the detected GFP signal (in arbitrary units). Below: a number of representative images of the microsieve. The time points at which the images were taken are indicated with an asterisk.

Figure 5. Co-cultivation of cells in the flow device.

Expression of GFP increases over time in cells growing in microdish wells after adding inducer (Acyl homoserine lactone (AHL) producing) and RFP expressing cells to the bottom compartment of the flow device. The intensity of the light emitted from five wells was quantified using imageJ and normalized against the background. No RFP was detected in the top chamber, indicating that the inducer-cells added to the bottom chamber did not come in contact with the top, and GFP expression was induced by diffusion of the inducer (AHL) through the microdish.

Figure 6. Fluorescent nematodes observed in the flow device.

A) Nematodes floating over the wells while the chamber is filled with liquid. The fluorescent oesophagus in the front side of the nematode is clearly visible. B) Nematode trapped in a well filled with fluorescent E. coli cells after removing the liquid from the chamber. C) Next day: A nematode after consuming all fluorescent bacteria from the well, resulting in observable fluorescence in the nematode intestine.