Automating the classification of single E. coli cells

The size and shape of bacteria can be revealing of cellular viability and health,1 similar to eukaryotic cells. Also, morphological changes can be suggestive of susceptibility or resistance to antibiotic treatment 2,3 and could be linked directly with genetic changes for functional genetic studies.4

Intrinsically, there is growing appeal to applying real-time microscopy for the development of antimicrobial susceptibility testing (AST).5 Furthermore, morphology linked to direct visualization of intra-bacterial structures might streamline studies of the mechanism of action in bacterial toxicity via direct visualization.

Imaging of vancomycin, for instance, was directly demonstrated to bind sites of peptidoglycan synthesis6 as it disrupts cell wall synthesis in Gram-positive bacteria.

The small size of the bacterium (approximately 1 mm) places several demands on the imaging system that has been utilized to obtain imaging data in an automated fashion. High magnification objectives of 40×–60× are needed.

The narrow depth of field characterizing these optics necessitates that the autofocus dependably places the cells on the surface of the substrate in the focal plane of the objective. This is to guarantee the capture of a sharp and high-contrast image.

Finally, screening studies including time-lapse imaging to catch dynamics require accurate sample movement to image the same cells over a period of time.

The Hermes® HCS imaging system was utilized here to obtain time-lapse images at 60x magnification, as displayed in Figure 1 and Figure 2.

Figure 1. Example time-lapse images (cropped) of E. coli cells at 60× in 15 minute intervals for 1 hour. Image Credit: IDEA Bio-Medical Ltd.

Automating the classification of single E. coli cells

Figure 2. Sub-population definition and growth-time curves. A) The cells are imaged at 60x magnification, segmented for identification and separated into populations. Oval-shaped cells (blue) have high axial ratios and high solidity, longer and more elliptical cells are in yellow. B&C) The time-plots of the general population (B, yellow) and Oval subpopulation (C), which are generated automatically within the Athena®, depict population changes over time. Image Credit: IDEA Bio-Medical Ltd.

Image analysis was executed with Athena® software with the help of the Cell Morphology application to count the number of E. coli cells present on the substrate surface and measure their populations depending on the shape.

Assay workflow

E. coli bacteria were grown to an OD of 0.6 in LB media to quantify whether they were in the exponential growth phase. Small samples were withdrawn and mixed with FM 4-64 membrane staining dye.

This labels the outer membrane of the E. coli cells. Furthermore, the labeled cells were diluted and positioned into numerous wells of an 8-well Ibidi chamber slide consisting of a thin glass bottom.

The imaging of the slide sample was done at 60× with one acquisition every 5 minutes for around 2 hours.

The images were then loaded into Athena® software for single-cell identification and counting. Cells consisting of smaller and more roundish shapes were separated from the ensemble with the help of the Athena’s® subpopulation tool along with two morphological attributes: solidity and axial ratio.

In this way, the growth data of bacteria consisting of various shapes can be plotted readily and compared against one another. Such analysis is of huge interest due to the presence of cell shape and its prevalence within a population is indicative of proliferative capacity and full viability.

Results and conclusion

E. coli cells that appear to be elliptical and longer are the representative of growing cells at the time of the exponential phase, whereas they are shorter and more ovoid and compact at the time of the stationary phase.7

Figure 2 portrays that even though the bacteria population increases with respect to time, the comparative proportion of the population comprising small and ovoid cells reduces with time (Figure 2C).

Such population decrease seems to be compatible with exponential growth and changes in its prevalence could apply to the identification of novel compounds that induce a stationary phase and avoid exponential growth.

E. Coli imaged on the WiScan® Hermes for 2 hours at 60x

E. Coli imaged on the WiScan® Hermes for 2 hours at 60×. Video Credit: IDEA Bio-Medical Ltd.


Cell preparation and data courtesy of Rafaël Sibilo and Professor Valerio Pruneri, Institut de Ciencies Fotoniques (ICFO), Castelldefels, Barcelona, Spain.


  1. Cabeen, M.T. & Jacobs-Wagner, C. Nature Reviews Microbiology, 3, (2005) pp 601-610. DOI: 10.1038/nrmicro1205
  2. Fredborg, M., Rosenvinge, F.S., Spillum, E. Kroghsbo, S., Wang, M., Sondergaard, T.E. BMC Microbiology 15, (2015)
  3. Lin T-Y, Gross WS, Auer GK, Weibel DB. mBio 10, (2019), e02401-18 DOI: 10.1128/mBio.02401-18 [4]
  4. French, S., Côté, J-P, Stokes, J.M, Truant, R., Brown, E.D. mBio 8, (2017), e01977-16.
  5. Belkum, A.v., Dunne, W.M. Journal of Clinical Microbiology, 51, (2013) pp 2018-2024
  6. Daniel, R.A., Errington, Jcell Therapy 113, (2003) pp 767-776.
  7. Deforet, M., Ditmarsch, D.v., Xavier, J.B. Biophysical Journal 109, (2015), pp 521-528

About IDEA Bio-Medical Ltd.

IDEA Bio-Medical is founded in 2007 through a partnership between YEDA (the Weizmann Institute’s commercialization arm) and IDEA Machine Development (an innovation hub).

We specialize in automated imaging systems and image analysis software, offering a broad range of biological applications based on the company’s unique algorithms library. The company is developing novel image-based screening platforms for the pharmaceutical industry and medical centers, dedicated to broadening the scope of personalized medicine.

Our WiScan Hermes system incorporates the most advanced technologies currently available in the machine vision field, integrated with engineering methodologies of high reliability and quality at the level of semi-conductors and digital printing industries, which are the specialty of our mother company, IDEA Machine Development Design and Production Ltd.

Sponsored Content Policy: publishes articles and related content that may be derived from sources where we have existing commercial relationships, provided such content adds value to the core editorial ethos of News-Medical.Net which is to educate and inform site visitors interested in medical research, science, medical devices and treatments.

Last updated: Jul 1, 2022 at 6:17 AM


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