Watch tutorials, webinars and informative videos about PreSens optical sensor systems.
Imaging the Oxygen Consumption of Microbial Cultures
Metabolic activity of E. coli monitored inside the incubator with VisiSens A1
H. Tschiersch1, G. Liebsch2, L. Borisjuk1, A. Stangelmayer2, H. Rolletschek1
1Department of Molecular Genetics, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, Germany
2PreSens Precision Sensing GmbH, Regensburg, Germany
Oxygen imaging with VisiSens™ A1 allows 2-dimensional mapping of oxygen consumption inside microbial culture. The compact measurement system can be applied for monitoring the metabolic activity directly inside the incubator. As an example, Escherichia coli cultures on agar were investigated to demonstrate the capabilities of the imaging device for visualizing oxygen gradients across the culture surface. The results show clearly how oxygen gradients evolve around single cultures and how oxygen levels decrease depending on the distance to other cultures over time.
Microbial and cell / tissue cultivations play a major role and are important tools in biological and medical research, or biotechnology. Knowledge on local metabolic activity of cells and structures within colonies may assist in understanding and controlling complex growth processes. One key parameter in cultivation control is oxygen. Oxygen consumption or production can give valuable information about the metabolic status of a culture. Precise oxygen measurement systems are therefore indispensable. However, conventional measurement techniques, like e. g. electrodes, only allow single-point measurements, lacking the spatial information about oxygen distributions in the sample. Even conducting transect measurements with oxygen microsensors cannot completely overcome this limitation. The VisiSens™ A1 system combines planar fluorescent sensor foils with digital camera technology, and now allows 2-dimensional assessment of oxygen distributions across the sample surfaces. The fluorescent sensor foils are non-toxic and do not consume oxygen during the measurement process. Information over a whole area can be recorded with µm resolution for extended measurement periods. The sensor foils are read out contactless with a compact fluorescence microscope. It can be easily installed inside an incubator and is controlled from the outside by PC or notebook (Fig. 1). Being able to visualize and evaluate changes in oxygen partial pressure due to metabolic and diffusion processes makes VisiSens™ the ideal tool for cell and tissue culture monitoring. We tested this measurement technology investigating oxygen distributions in different plant tissues, and also tried it in a non-plant application. In the experiment described here E. coli was used as a model microorganism. The culture was monitored with the VisiSens™ A1 system inside the incubator, and the oxygen consumption of single bacteria colonies was visualized.
Materials & Methods
At the start and the end of the experiment the VisiSens™ oxygen sensor foil (SF-RPSu4, PreSens, Regensburg) was calibrated by recording images of both a sodium sulfite solution (0 % air saturation) and of air saturated distilled water (100 % air saturation). E. coli colonies were grown on an agar plate and the sensor foil was put directly on the culture. The VisiSens™ detector unit (DU01, PreSens) was installed inside the incubator and controlled from the outside with a laptop. Detector unit control and subsequent image analysis were realized with the VisiSens™ AnalytiCal 1 software. Image recording was started immediately after the culture was put in the incubator for an incubation period of 20 minutes; images were taken at an interval of 30 s.
Imaging E. coli Colonies
Short-term incubation (5 - 20 min) with the oxygen sensor resulted in clear oxygen distribution images, where single colonies were identifiable by their oxygen consumption (Fig. 3). The oxygen concentration at the location of the colonies decreased rapidly within 20 min (Fig. 4 + 5). The oxygen images further reveal how the oxygen decrease depends on the distance to other colonies (Fig. 4). Driven by diffusion and therefore by oxygen consumption of multiple neighboring E. coli colonies the oxygen level inside the medium also changes over time (Fig. 6).
VisiSens™ is able to visualize different sites within a multiwell plate or Petri dish. Applied in microbial culture it is possible to distinguish oxygen consumption of single colonies. By visualizing oxygen distributions across the sample and its changes over time valuable information can be gained, which can be used to monitor the metabolic status or to modulate oxygen supply to better control certain processes in cell or tissue culture. The use of VisiSens™ facilitates the study of respiratory dynamics and will contribute to a better understanding in many application fields, like e. g. the screening for respiratory phenotypes in yeast.