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 This article contains a small extract from Chapter 7 of “Atomic Force Microscopy”. Chapter 7 contains descriptions of applications of AFM in materials science, chemistry and physics, biology and the life sciences, nanotechnology, and in industry. This short section describes some examples of applications of AFM in bacteriology. References lists, and the second figure can be found in the full book.

AFM is a highly suitable tool to examine bacteria, and has been widely applied to their study. Bacteria are commonly studied by optical microscopy, which can give an overall idea about gross cell morphology (via a two-dimensional projection), and is also useful for cell-counting studies. In comparison, AFM is slower, and thus is less useful for quantitative cell-counting, but allows measurement of a variety of other cellular properties, particularly by nanoindentation and force spectroscopy experiments [611]. In addition, the greatly increased resolution of AFM allows for the imaging of finer details of cell morphology and sub-cellular features such as pili and fimbriae [612]. The three dimensional information from AFM can also be useful in differentiating morphologies which would look the same in optical microscopy [6]. Various other micro-organisms have been studied by AFM such as spores [178, 613–615], fungi [616, 617], including yeasts [171, 618], viruses [287, 619], and others [620] but here we concentrate on bacteria for the sake of brevity.

fig 7.20

Fig. 7.20. Studies of bacterial morphology. Top left: Streptococcus, showing typical linear clusters. Top right: large clusters of Staphylococcus aureus. Bottom left: Salmonella biofilm showing pili-like fimbrial structures. Bottom right: E. coli. All these images were measured in air. Reproduced with permission from [624] (top left) and [626] (bottom left). Right hand images the author's own work.

Some species of bacteria that have been well-studied include E. coli [169, 621] and various species of Staphylococcus [169, 317, 622], Bacillus [178, 615], Streptococcus [623, 624] and Salmonella [625, 626], see Figure 7.20. Bacteria generally need to be immobilized on a surface for imaging, and a number of different procedures have been used. For studies in air, drying onto a surface, or even flaming can work well, although one needs to be careful of drying artefacts by these techniques [621]. However, it is useful to be able to study bacteria in liquid, and usually this requires a more elaborate preparation protocol because it’s necessary that the cells be fixed to the substrate in some way. The most commonly used techniques include the use of poly-l-lysine (PLL) or polyethyleneimine (PEI) coating of glass for chemical capture and using gelatin-coated glass for a soft physical capture [6, 314, 315]. For spherical bacteria, i.e. cocci, physical trapping of substrates with appropriately-sized holes works well, and it can even be possible to observe the cells dividing while immobilized in this way [313, 317]. Bacteria that naturally form biofilms are simple to study as biofilms are perfect samples for AFM, although some washing may be required. See Section 4.1 for more sample preparation details. Although higher resolution is usually obtained in air, bacteria imaged in liquid are closer to the native state, and dried bacteria usually have a small fraction of their hydrated height [6, 315, 621].

One of the most important areas in studies of bacteria is the study of the method of action of antibiotics and other antibacterial agents, due to the ongoing increase in antibiotic resistance in bacteria [627]. Several studies have imaged bacteria treated with antimicrobial agents, including the morphological changes to E. coli caused by the antibiotic cefodizime [628] and also E. coli and P. aeruginosa response to antibacterial peptides [629, 630], S. aureus response to antibiotics [631, 632] and others [624, 633, 634]. The response to the natural antimicrobial polymer chitosan, of E. coli, S. aureus, B. cereus and B. cereus spores has been measured by both AFM imaging and nanoindentation measurements [169, 178]. The changes that can be seen include morphological alterations such as appearance of holes, shrinking, cell shape changes and cell lysis, and also mechanical changes. An example showing the response of S. aureus to antibiotic treatment by both topographic changes and changes in cell elasticity is shown in Figure 7.21.

[Figure 7.21 available in the full book]

It’s also useful to make measurements of bacteria by non-imaging modes of AFM, because the high positioning resolution of AFM allows such measurements to directly address individual bacterial cells, which is difficult by other techniques [611]. For example, nanomechanical measurements (e.g. nanoindentation ) of bacteria have been

 

shown to be sensitive to treatment with antimicrobial agents [169, 629, 632], bacterial species and strain [155, 635, 636], physiological state of the organisms and the environment in which the measurements are made [155]. With the AFM it’s relatively simple to perform nanoindentation experiments on individual micro-organisms, and even to differentiate one part of a cell from another by stiffness measurements [171]. For this sort of experiment, it’s important to remember that the response of the probe will be different when the cell surface is perpendicular to the probe motion, than when it’s at an angle, however [637]. Thus, all measurements should normally be carried out only on the upper portion of the cell which is relatively flat [382]. Other non-imaging experiments which may be carried out on bacteria using AFM include force spectroscopy in order to measure the distribution of specific adhesion factors on cell surfaces [156], cell hydrophobicity/hydrophilicity [475, 638], or the distribution of other molecules across the cell surface [611, 637, 639].

Bacterial colonization of surfaces is an important process, and reducing the process requires knowledge of individual bacteria–surface interactions. Bacteria–surface adhesion studies can be carried out using a number of experimental methodologies, the most commonly applied ones being direct force spectroscopy with bacteria immobilized on the AFM probe and lateral force microscopy measurements of the force required for removal of cells [637, 640–644]. AFM allows the combination of studies of cell–surface adhesion, with measurements of the surface itself, which can help to understand how factors such as roughness, hydrophobicity, etc. can affect colonization by bacteria [645].

 


 

This text, and the compiled figure is copyright 2010 by Peter Eaton. The copyright for the indivudual figures in the compiled image rests with the journals in which they were published:

[624] Braga, P. C. and Ricci, D., Differences in the susceptibility of Streptococcus pyogenes to rokitamycin and erythromycin A revealed by morphostructural atomic force microscopy. Journal of Antimicrobial Chemotherapy 2002, 50 (4), 457–60.

[626] Jonas, K.; Tomenius, H.; Kader, A.; Normark, S.; Romling, U.; Belova, L.; Melefors, O., Roles of curli, cellulose and BapA in Salmonella biofilm morphology studied by atomic force microscopy. BMC Microbiology 2007, 7, 70.

[My Work]: The S. aureus image appeared in: P. Eaton, et al., Atomic Force Microscopy Study of the Antibacterial Effects of Chitosans on Escherichia coli and Staphylococcus aureus, Ultramicroscopy 108(10), 1128-1134 (2008). The E. coli image is unpublished, but Copyright 2009 Peter Eaton.

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