Elsevier

Ultramicroscopy

Volume 154, July 2015, Pages 29-36
Ultramicroscopy

Nanoscale imaging of the growth and division of bacterial cells on planar substrates with the atomic force microscope

https://doi.org/10.1016/j.ultramic.2015.02.018Get rights and content

Highlights

  • Gelatine coatings used to weakly attach bacterial cells onto planar substrates.

  • Use of the dynamic jumping mode as a non-perturbing bacterial imaging mode.

  • Nanoscale resolution imaging of unperturbed single living bacterial cells.

  • Growth and division of single bacteria cells on planar substrates observed.

Abstract

With the use of the atomic force microscope (AFM), the Nanomicrobiology field has advanced drastically. Due to the complexity of imaging living bacterial processes in their natural growing environments, improvements have come to a standstill. Here we show the in situ nanoscale imaging of the growth and division of single bacterial cells on planar substrates with the atomic force microscope. To achieve this, we minimized the lateral shear forces responsible for the detachment of weakly adsorbed bacteria on planar substrates with the use of the so called dynamic jumping mode with very soft cantilever probes. With this approach, gentle imaging conditions can be maintained for long periods of time, enabling the continuous imaging of the bacterial cell growth and division, even on planar substrates. Present results offer the possibility to observe living processes of untrapped bacteria weakly attached to planar substrates.

Introduction

Since the first images of dried bacterial cells were obtained with the Atomic Force Microscope (AFM) [1], this technique has significantly contributed to the understanding of the nanoscale structural and physical properties of single bacterial cells [2], [3], [4], [5], [6]. Examples include the high resolution imaging of the dynamics of bacterial membrane proteins [7], [8], the molecular recognition of cellular membrane proteins [9], [10], the visualization of the effects of antibiotics on the cell surface [11], [12], and imaging of the extrusion of bacteriophages [13]. In this way, the AFM has decisively contributed to the emerging field of Nanomicrobiology [5].

Imaging living bacterial cells with the Atomic Force Microscope still poses a major challenge. This limitation arises from the relatively reduced adsorption forces of most living bacteria to the standard substrates used for AFM (such as glass or mica). In contraposition, the non-living bacterial cells (i.e dried bacteria) show stronger adhesion forces, making imaging easier and extensively used [14], [15].

Two different approaches have been reported to overcome the difficulty of imaging living bacteria. The first approach relies on increasing the strength of the forces that immobilize the bacteria to the substrates. The second approach is focused to reduce the shear forces exerted by the AFM tip on the bacteria and which are responsible for cell detachment during imaging. Among the first approach, we can find the physical entrapment of bacterial cells into polycarbonate filters [8], [16] or microwells [17], or the use of specific substrate coatings (such as APTES [11], PEI [18], poly-L-Lysine [19], [20], polyphenolic proteins [21] or gelatine [21], [22], [23]) or surface chemical binding groups (e.g. cross-linking of NH2 groups via glutaraldehyde [24]). Concerning AFM imaging modes, conventional modes such as contact mode or dynamic mode can only be used when bacteria are relatively strongly attached to the substrates [25]. For weakly attached bacteria (for most coated planar substrates) the use of the intermittent contact mode with magnetically excited probes seems to offer the best performance [17], [19], [22]. This has been attributed to the fine tuning of the dynamic oscillation in liquid conditions.

Despite these developments, relatively little progress has been made in the nanoscale imaging of living bacterial processes, such as bacterial growth and division [16], [17], specially for bacterial cells on planar substrates [19], [26]. The use of planar substrates provides a more natural condition to study these bacterial processes. They offer a less constrained space (compared to physical entrapment methods) for bacterial growth and division, together with weak electrostatic adsorption forces. In this way, it mimics the bacterial natural way of adhesion onto several types of substrates, including those present in biofilm formation on natural and synthetic surfaces [27], [28]. In this paper, we present the use of an alternative AFM imaging mode to study living bacterial cells, the so called dynamic jumping mode. With this method, we have been able to image living bacterial cells weakly absorbed onto planar substrates, following its growth and division. When using dynamic jumping mode, the probe is oscillated at its resonance frequency and approached to the sample until a prefixed oscillation amplitude set point is reached. At this point, the probe is retracted a given distance and laterally displaced out of contact from the sample until the next point. This out of contact lateral displacement, together with the use of the intermittent contact mode and of soft probes, drastically reduces the shear forces exerted onto the weakly absorbed bacterial cells. It should be noted that dynamic jumping mode offers a better performance than its static version [29], which has already been widely used in the imaging of viruses on planar substrates in physiological conditions [30], [31].

With the use of the dynamic jumping mode we have been able to image living single bacterial cells belonging to two different Escherichia coli strains, the MG1655 and the enteroaggregative (EAEC) 042, both being weakly adsorbed onto planar gelatine coated substrates. In addition, we have been able to monitor the growth and division of E. coli 042 in its native state over long periods of time.

Section snippets

Cell types and cultures

E. coli strain MG1655 is well known to be the common non-pathogenic laboratory E. coli strain for biological research [32], while strain 042 is the archetype of the EAEC pathotype [33], [34], [35]. EAEC strains display a characteristic aggregative or ‘‘stacked-brick’’ pattern of adherence to intestinal epithelial cells [36]. When grown at initial stages of biofilm, bacteria secrete less extracellular polymeric substance (EPS) [37].

Stock samples of the common laboratory strain E. coli MG1655 and

Imaging bacterial cells on planar substrates in buffer solution

For further reference, we started the analysis by analyzing the E. coli 042 strain grown according to protocol 1 in both dry and re-hydrated conditions. Fig. 1A shows an image obtained under nitrogen ambient flow (~0% Relative Humidity) of a dried (and hence dead) bacterial cell on a gelatinized gold substrate. Dried cells presented a rod-shaped structure ~2 μm long and ~1 μm wide and with a maximum height ~261±6 nm (N=13), as obtained from cross-sectional profiles taken along the main bacterial

Discussion

We have shown that the dynamic jumping mode implemented with soft cantilevers enables the nanoscale AFM imaging of viable and metabolically active bacteria on planar substrates. The use of weak forces (lower than 0.2 nN), together with the lateral displacement of the probe far away from the sample (which drastically reduces lateral shear forces) are at the basis of this capability. Based on the results obtained, this mode can be considered as an alternative to other existing AFM imaging modes

Conclusions

We have shown that dynamic jumping mode AFM constitutes a powerful technique for the observation of physiological processes of viable bacteria that are weakly attached to biocompatible gelatinous coated planar substrates. Images of intact and viable bacterial cells have been obtained for cells suspended in buffer solution for two different E. coli bacterial strains on different substrates, thus predicting a wide applicability of this imaging method. We have observed that when imaging in

Acknowledgments

This research has been financially supported by the Spanish Ministry of Education and Science under Grant no. TEC2010-16844, and by the European Commission under Grant no. NMP-280516. We acknowledge T. Wiegand for useful suggestions.

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