Morphological and mechanical imaging of Bacillus cereus spore formation at the nanoscale

Journal of Microscopy, Vol. 258, Issue 1 2015, pp. 49–58 doi: 10.1111/jmi.12214

Received 8 September 2014; accepted 11 December 2014

Morphological and mechanical imaging of Bacillus cereus sporeformation at the nanoscale

C O N G Z H O U W A N G ∗, C R I S T I N A S T A N C I U†, C H R I S T O P H E R J . E H R H A R D T† &V A M S I K . Y A D A V A L L I ∗∗Department of Chemical and Life Science Engineering, Virginia Commonwealth University, Richmond, Virginia, 23284, U.S.A.

†Department of Forensic Science, Virginia Commonwealth University, Richmond, Virginia, 23284, U.S.A.

Key words. Atomic force microscopy, Bacillus, nanoindentation, sporulation.

Summary

Bacteria from the genus Bacillus are able to transform intometabolically dormant states called (endo) spores in responseto nutrient deprivation and other harsh conditions. Thesemorphologically distinct spores are fascinating constructs,amongst the most durable cells in nature, and have attractedattention owing to their relevance in food-related illnessesand bioterrorism. Observing the course of bacterial spore for-mation (sporulation) spatially, temporally and mechanically,from the vegetative cell to a mature spore, is critical for abetter understanding of this process. Here, we present a fastand versatile strategy for monitoring both the morphologi-cal and mechanical changes of Bacillus cereus bacteria at thenanoscale using atomic force microscopy. Through a strat-egy of imaging and nanomechanical mapping, we show themorphogenesis of the endospore and released mature en-dospore. Finally, we investigate individual spores to charac-terize their surface mechanically. The progression in elasticitycoupled with a similarity of characteristic distributions be-tween the incipient endospores and the formed spores showthese distinct stages. Taken together, our data demonstratesthe power of atomic force microscopy applied in microbiol-ogy for probing this important biological process at the singlecell scale.

Bacteria of the genus Bacillus are rod-shaped, Gram-positivecells with the ability to metabolically transform into oval, dor-mant cells called endospores in response to nutrient depriva-tion conditions (Errington, 1993; Piggot & Hilbert, 2004). Theprocess of forming the bacterial endospore is called sporula-tion, and is directed by a series of complex, tightly regulatedgenetic programs. During this process, each vegetative cellgenerates one endospore and the mother cell undergoes lysis

Correspondence to: Vamsi K. Yadavalli, Department of Chemical and Life Science En-

gineering, 601 W. Main Street, Virginia Commonwealth University, Richmond, Vir-

ginia, U.S.A. Tel: 804-828-0587; fax: 804-827-3046; e-mail: [email protected]

as the mature endospore is released (Kuo et al., 2006). Thespore formed is quite distinct from the vegetative cell with newsynthetic protective layers, which causes it to be extremelyresilient and resistant to harsh external environmental con-ditions including pH, temperature and nutrient deficiency(Setlow, 1994; Pinzon-Arango et al., 2010). Although thespore is metabolically inactive, it maintains the ability tomonitor its environment and can transition back to vegeta-tive growth once exposed to nutrients (germination; Setlow,2003). Understanding and observing the process of sporula-tion, in particular the onset of endospore formation, has greatfundamental and applied importance (Hauser & Errington,1995; Barak et al., 2005; Tan & Ramamurthi, 2014). Sporeforming bacteria such as Bacillus anthracis are important as thecausative agents of anthrax. Bacillus cereus, a typical Gram-positive spore-forming bacterium, is a common food contami-nant and able to cause food poisoning and infections (Liu et al.,2007). The thermo-resistant B. cereus spore is able to surviveduring food processing and preservation processes, which cre-ates a potential safety problem for the food industry (Arnesenet al., 2007). Here, the tough and protective structure of thespore plays an important role in resistance to external envi-ronmental stresses and inhospitable conditions (Henriques &Moran, 2000).

There are unique differences in the spore’s cellular struc-tures in comparison to the vegetative state (Higgins &Dworkin, 2012). Morphological changes during sporulationare accompanied by variations in the mechanical properties ofthe cell surface as multilayers of protein coats and peptidogly-can layers encase cellular contents and genetic informationinto the newly formed spore (Aronson & Fitzjames, 1976). Todate, several approaches have been applied to observe sporu-lation, primarily involving optical and electron microscopy,(Manasherob et al., 1998; Ghosh et al., 2009; Tocheva et al.,2013) and a fairly detailed view of this transition is now avail-able (Driks, 2002; Higgins & Dworkin, 2012). However, typ-ically these methods either have low spatial resolution, orrequire complex sample preparation including spore-specificstaining for optical or fluorescence microscopy, or ultrathin

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sections necessary for transmission electron microscopy. Im-portantly, these techniques are unable to directly visualize themorphological changes on the bacterial cell surface or, in thecase of electron microscopy, observe real time changes undernear physiological conditions. Further, it is difficult to applyexisting technologies to study sporulation in complex environ-mental samples such as biofilms. The development of a simple,fast and versatile platform to monitor in situ bacterial sporula-tion from a morphological and mechanical perspective at thenanoscale can provide key insights into this process.

Atomic force microscopy (AFM) has rapidly emerged as animportant, widely used tool in microbiology (Dufrene, 2008;Dupres et al., 2010; Muller & Dufrene, 2011). The uniqueadvantage of the AFM is the ability not only to characterizecellular surfaces with nanoscale resolution and three dimen-sional imaging (Plomp et al., 2007), but also measure theirnanomechanical forces (Dufrene & Pelling, 2013). Samplesfor AFM do not require preparative steps such as dye staining,fluorescence labelling, conductive coating or vacuum, and canbe measured under near physiological environments (Fernan-des et al., 2009; Gillis et al., 2012). The addition of mechanicalcharacterization capabilities to the AFM toolkit offers a newway to observe surfaces of a variety of hard and soft materials.Using the AFM tip as an indenter, quantitative informationon the elasticity of the sample can be obtained (Ebenstein &Pruitt, 2006). In recent years, the process of collecting suchdata has been further expanded with the advent of automatedscanning modes that allow us to rapidly obtain spatial distri-butions of elasticity (Kuznetsova et al., 2007; Alsteens et al.,2013). Recently, “multi-harmonic AFM” was applied to mapthe surface nanomechanical properties of Escherichia coli withhigh time and spatial resolution (Raman et al., 2011). Ap-plications of AFM based multiparametric mapping in cellularsystems have been covered in recent reviews (Dufrene et al.,2013). Therefore, both nanoscale morphology and elasticitychanges on live cell surfaces can be monitored in real time.Over the past decade, there have been several AFM studies onbacterial spores including high-resolution nanoscale imaging,(Chada et al., 2003; Plomp et al., 2005b; Kailas et al., 2011)observing how antibacterial ingredients or harsh conditionsaffect the morphology and mechanical properties, (Kuo et al.,2006; Fernandes et al., 2009; Xing et al., 2014) and the effectof nutrients on the morphology and mechanical propertiesof spores during germination (Pinzon-Arango et al., 2009;Pinzon-Arango et al., 2010). These reports have shown bothmorphological and mechanical analyses of Bacillus spores viaAFM at the nanoscale (Touhami et al., 2003a). However, di-rectly following the nanomechanical changes of the vegetativecell during sporulation, particularly at the complete and singlecell level has not been shown.

In this work, we present a novel application of nanoscaleAFM imaging and nanomechanical mapping to observe thesporulation cycle of B. cereus at the single cell level. Inaddition to observations on the altering of cell shape and

morphology, we show, by probing the cell surface that stagesof sporulation may be observed by observation of the mechani-cal nature of the cell. We selected B. cereus as a model organismto explore this process. B. cereus further shares a genetic andstructural similarity with the more virulent B. anthracis (Readet al., 2003; Abdou et al., 2007), a select agent of interest to thebiodefense community. Using AFM, the morphology and me-chanics of B. cereus at different time points during sporulationwere imaged. These images, for the first time, clearly showthe distinctive stages of the morphogenesis of spores duringsporulation from a mechanical perspective. Using nanoinden-tation to characterize the surfaces of cells at different stagesand “whole cell elasticity maps” obtained provide a uniqueglimpse into the cell-spore progression and correlate well withelasticity measurements on mature spores. These results detailhigh-resolution AFM imaging coupled with nanoindentationas a versatile platform to quantitatively monitor the changesof morphology and mechanical properties during bacterialsporulation at the nanoscale.

Materials and methods

Sample preparation and sporulation conditions

Cultures of B. cereus (T-strain) were maintained at 30°C onTrypticase Soy Agar (30 g Trypticase soy broth, (BectonDickinson, 211768, Franklin Lakes, NJ, USA) and 15 gagar (American BioAnalytical, AB1185, Natick, MA, USA)).Starter cultures were grown by inoculating single colonies ofB. cereus into 125 mL of Trypticase soy broth and incubatingfor 24 h at 30°C and 225 rpm. G Medium was used as the basesporulation formulation for all recipes (Hornstra et al., 2006).Sporulation was performed by adding 1 mL of starter cultureinto 250 mL of sporulation medium and incubated at 30°Cand 275 rpm in an orbital shaker. Six different time pointsduring sporulation were selected to make samples: 0, 8, 12,16, 24 and 42 h. ‘0 h’ represents the B. cereus in starter culturebefore triggering sporulation. The others represent the cells insporulation conditions (culture in G medium).

AFM imaging

For each sample, a 1 mL cell suspension was taken from theculture medium and placed into a sterile 2 mL Eppendorftube. Cells were collected by low speed centrifugation (1 min,4°C, 4000 rpm). Each pellet was resuspended and washedtwo times with 2 mL sterile distilled water. Finally, the pelletwas resuspended in 2 mL sterile distilled water and 10 μL cellsuspension was spotted onto a clean glass slide and dried inair. AFM imaging was performed in noncontact mode usingan Asylum MFP-3D atomic force microscope (AFM, AsylumResearch, Santa Barbara, CA, USA). Cells were imaged in airto clearly show surface topography features, including surfacenanostructures that may be obscured in imaging under liquid

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environments (Chao & Zhang, 2011; Gillis et al., 2012).Super sharp silicon cantilevers (AC240TS, spring constant�2 N m−1, resonance frequency 70 kHz) from Olympus(Tokyo, Japan) with nominal radii of curvature 9 ± 2 nm wereused for imaging samples at a high resolution. The morphologyof these samples including height profile and cell size were anal-ysed using custom routines in Igor Pro 6.32 (Wavemetrics Inc,OR, USA).

Measurement of mechanical properties via nanoindentation

AFM-based nanoindentation experiments were carried outusing different probes(k � 20 N/m) to those used for imaging(PPP-ZEIHR, Nanosensors, Neuchatel, Switzerland). Springconstants of cantilevers were measured prior to each ex-periment using the thermal fluctuation method (Hutter &Bechhoefer, 1993). Stiffer cantilevers were used owing to thenature of the cells probed in air, with well-known high rigidityof bacterial spores (Fernandes et al., 2009). In contrast to con-ventional point-by-point load-indentation reported earlier, astrategy of elasticity mapping was used for these experimentsto observe the entire cell area. Indent curves were obtainedby collecting a series of sequential indent curves in an m × ngrid. Each indent curve was obtained at the same loading rate(300 nN s−1) by indenting the cantilever to the samples until aconstant load force (150 nN) was reached. All the proceduresincluding analysis of the indent curves using the Hertz model,(Touhami et al., 2003b; Lin & Horkay, 2008) generating elas-ticity maps as well as the overlays of height maps andelasticitymaps were carried out with Igor Pro 6.32 software. Elasticitymaps were obtained by collecting 50 × 50 indent curves over adefined area (�3 × 3 μm) and estimating the Young’s moduli.

Results and discussion

The complex development of Bacillus endospores allows themto respond to (and survive) challenging conditions. Elucidat-ing the process of spore formation therefore has importantimplications for Bacillus ecology, pathogenicity and environ-mental persistence. Typically, endospores have been identifiedusing techniques such as phase contrast microscopy, whereinthey can appear edged and bright, fluorescence or light mi-croscopy (Tocheva et al., 2013), wherein they may react withstaining reagents, or using electron microscopy (Bulla et al.,1969) wherein an extra conductive coating is needed on sam-ple surfaces (Todar, 2009). The AFM provides a way to studysingle cells using topographical measurements in 3D space. Inthis study, we focus on the use of the AFM to investigate thenanoscale morphological and nanomechanical properties ofB. cereus through the earliest stages of spore formation withinthe mother cell through its release into the environment.

Fig. 1. Corresponding optical phase contrast images (left, scale bar =5 μm) and AFM topography images (right, scale bar = 1 μm) of Bacilluscereus before and after sporulation was triggered. (A): 0 h. (B): 8 h. (C):18 h. (D): 24 h. ‘18 h’ is the earliest time point when endospores canbe observed (bright spots). Note that the z-scale is the same on all AFMimages.

Morphological observation of sporulation at the micro andnanoscale

AFM based techniques have been previously applied forspecifically characterizing bacterial processes (Liu & Wang,2010). Observing the time-resolved sporulation process at ahigh resolution using AFM imaging involves proper imagingenvironments and sample preparation. Initial results showed


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Fig. 2. Topography (top row) and AFM amplitude (bottom row) images of Bacillus cereus before and at the onset of sporulation trigger. (A, D) Heightimage before cells were added to G medium. (B, E) Following an 8 h culture. (C) Following 12 h. The red arrows show the onset of sporulation. Scalebars = 1 μm.

that the bacterial images in liquid had lower resolution incomparison to imaging in air which showed refined details ofsurface morphology (Fig. S1). Thus, in these experiments, asimple air-drying method was used to fix the cell samples on asubstrate – a small volume of cell suspension was deposited ona glass surface. Following adsorption, the substrate was gentlyrinsed with sterile water to remove salts which might createartefacts on cell surfaces. As samples were extracted fromthe sporulation culture medium at certain time points, thisenables us to track the entire process in ‘real-time’ and observedistinct stages. Figure 1 shows the different stages of B. cereussporulation associated with phase contrast microscope imagesand AFM height images at different time points includingbefore and after sporulation was triggered. It is important tonote that sporulation times can vary greatly depending on theexternal environment (Higgins & Dworkin, 2012). In termsof time point and morphology, the phase contrast images canbe correlated with the AFM height images, wherein brightareas (endospore) can be observed on both images.

The nanoscale surface morphology of B. cereus cell surfacesat different time points is shown in Figures 2 and 3 asrepresentative AFM height and amplitude images on smallerareas of samples. As a starting point, the vegetative cells priorto addition to G medium (‘0 h’) were observed to be rod-shapedand forming vegetative cell chains (Fig. 2A). A quantitativemorphology analysis was performed on several cell samplesfrom different areas and images (n = 20). The dimensions of

these vegetative cells were 2.8 ± 0.5 μm in length, 1.2 ±0.2 μm in width, consistent with previously reported dimen-sions (Pan et al., 2006; Fernandes et al., 2009). Height profileswere measured along the ‘long-axis’ of single cells as the cellsurface was observed to change in flatness over time, especiallyas the endospores develop. Three morphology parameterswere analysed from height images: Hmax, the maximal heightof single cells, �H, the maximal height difference on singlecell surfaces, wherein the height of the endospore could beestimated as the difference in the heights between the top andthe flat region of the cell, and the root mean square roughnesson single cell surfaces (Fig. 4A). At the ‘0 h’ stage, the maximalheight of single cells is 366.5 ± 12.2 nm, consistent with re-ported values also measured by AFM (Fernandes et al., 2009).No obvious bumps are observed on the height and amplitudeimages coupled with 34.3 ± 3.5 nm of the maximal heightdifference and 22.2 ± 5.6 nm of root mean square roughnesson single cell implies that the cell surfaces are flat and noendospores formed before the sporulation was triggered.Following an 8-h period, cell imaging showed no obviousdifferences compared to the 0 h vegetative cells (Fig. 2B),indicating that the endospore formation had not begun.

Figure 2(C) represents the onset of sporulation after 12 hin culture, wherein the cells were observed to have a raisedstructure at one end (red arrows). This is observed by vi-sual examination as well as an increase in the maximalheight of single cells and height differences from AFM imaging



Fig. 3. Topography (top row) and AFM amplitude (bottom row) images of Bacillus cereus after sporulation was triggered. (A, D) 18 h. (B, E) 24 h. (C, F)42 h. At this point, the cells lyse and spores are released. Red arrows show the exosporium of the released endospores. Green arrows show the surroundingcell debris. Scale bars = 1 μm.

Fig. 4. (A) RMS roughness values obtained from cell surfaces via AFM imaging (n � 10 cells). As spores form, the roughness of the cells increase till thecells are lysed at t = 42 h. (B) Schematic representation of morphology analysis associated with of a nanoindentation experiment on a ‘24 h’ sample.‘Hmax’ represents the ‘maximal height of single cell (nm)’. ‘�H’ represents the ‘maximal height difference on single cell surface’.

(Table 1). This morphology change shown at this time pointmay be caused by the asymmetric division of the protoplasm,which is the first morphological characteristic of sporulationprocess (Piggot & Hilbert, 2004). Figure 3 shows a rapid pro-gression of the sporulation process till the lysis of the cell andsubsequent release. In Figure 3(A), at 18 h, the endospore-likebumps are more pronounced and observable. This is consis-tent with the image from phase contrast microscope (Fig. 1C),wherein the endospores could be clearly observed at thistime point. Expectedly, the height profile analysis shows themaximal height of single cells, height difference and root mean

square roughness on cell surfaces increase significantly, im-plying that the endospores were building their thick and mul-tilayered spore walls (Fig. 4A; Chada et al., 2003; Tochevaet al., 2013). At these intermediate points, it is worth not-ing that the height difference on cell surfaces increased is dueto two reasons including the increase of maximal height incertain areas of the spores with a simultaneous decrease insurrounding areas. This also can be observed from the verythin and flat (�40 nm) edges of the mother cells (Fig. 3B).By contrast, vegetative cells are largely homogeneous inheight with a very small difference between the highest and


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Table 1. Morphology analysis of Bacillus cereus at different time points during sporulation. The height profile measurements were performed on severalimages on different areas (n = 20). A: Maximal height of single cells (nm). B: Maximal height difference on single cell surfaces (nm)

0 h 8 h 12 h 18 h 24 h 42 h

A 366.5 ± 12.2 370.3 ± 12.7 442.0 ± 50.9 667.5 ± 27.0 703.9 ± 22.8 789.6 ± 17.4B 34.3 ± 3.5 37.0 ± 5.2 183.4 ± 36.9 418.5 ± 41.5 585.2 ± 41.40 740.8 ± 35.2

Fig. 5. Change of nanoscale mechanical property before and after sporu-lation was triggered. (A) Elasticity map of vegetative cells. (B) Elasticitymap after cells were cultured in G medium for 24 h. (C) Elasticity map aftercells were cultured in G medium for 42 h. The scale bar in each imagerepresents 250 nm. Scan sizes were varied to capture entire cells.

lowest points of the cells. Finally, after 42 h, the isolated matureendospores are formed and released (Fig. 3C). Besides the char-acteristic ovoid shape of the spores, (Errington, 1993) severalmorphological characteristics confirm the spore formation –(i) surface ridges extending along the entire spore length, acharacteristic surface feature owing to the folding of the sporecoat in the dehydrated state (Plomp et al., 2005a; Chen et al.,2014). (ii) An ultrathin membrane (�20 nm of height) sur-rounding the spores (red arrows in Fig. 3C). This exosporium

is another characteristic feature of B. cereus spores (Ankolekar& Labbe, 2010). (iii) Debris on the substrate indicating lysis ofmother cell after the mature spore is released (Hosoya et al.,2007). (green arrows in Fig. 3C) and (iv) A significant lengthdecrease of cells. The spores with 1.8 ± 0.2 μm in length areshorter than that of initial vegetative cells (2.8 ± 0.5 μm).

Nanomechanical characterization of sporulation

In addition to morphology variation, measuring the mechani-cal properties of cellular systems is an important parameter tocharacterize various cellular processes including adhesion, di-vision, sporulation and carcinogenesis (Suresh, 2007; Dufrene& Pelling, 2013). There have been a few reports on mea-suring mechanical changes during cellular processes usingAFM based nanoindentation. These have included changesin Escherichia coli before and after thermal treatment, (Cerfet al., 2009) changes in stiffness of neurons during neuriteoutgrowth and upon disruption of microtubules, (Speddenet al., 2012) and the elasticity variation of Pseudomonas aerug-inosa prior to and after antibiotic treatment (Formosa et al.,2012).However, the transition of mechanical properties fromthe vegetative cell to the spore has not been reported. Ingeneral, the nanomechanical characterization of B. cereusis largely unknown. During the sporulation process, the B.cereus mother cells synthesize multiple protein layers (innercoat and outer coat) and a thick peptidoglycan layer (cortex)for building the endospore. This implies that endospore for-mation would be accompanied by a change in the elasticityof the cell surfaces. Also, we postulate that if the observedbump at either end of these cells is a real, incipient endospore,the elasticity of these structures should be close to that ofa mature endospore and different from the rest of the cell.The AFM allows us to determine this progression via nanoin-dentation coupled with a technique of ‘elasticity mapping’ todetermine the mechanical nature of the entire cell surface atthe nanoscale (Fig. 4B). Three representative samples at 0, 24,42 h and a mature spore were studied. The indent curves fitwell to a Hertzian mechanical model, allowing us to obtainspatially resolved Young’s moduli. In each case, the mechan-ical elasticity map could be overlaid on the topography of thecell to show the variation of properties across the entire cell(Fig. 5).

Initially, vegetative cells were probed as shown inFigure 5(A). The overlay of elasticity map and heightmap shows good correspondence between the elasticity and



Fig. 6. (A) Nanomechanical map of a Bacillus cereus cell after 24 h of initiation of sporulation. The underlying spore is clearly visible from the topographyand elasticity maps. (B) The elasticity map (50 × 50, higher resolution on smaller area) corresponds to the area (marked by red box) in the height map.

morphology (softer on vegetative cell surface and stiffer onglass). The average Young’s modulus across the vegetativecell surface is 0.72 ± 0.29 GPa, which is in excellent agree-ment with the reported value of 0.83 ± 0.48 GPa measured ata few points on a B. cereus cell surface (Fernandes et al., 2009).Following sporulation and the development of the nascentendospore, three characteristic regions of elasticity can be ob-served on the cell surface (Fig. 5B, sample after ‘24 h’ sporu-lation): (i) an area on the flat surface of mother cell with anaverage modulus (2.52 ± 0.59 GPa) that is higher than thatof a vegetative cell. (ii) A softer ‘ring-like’ area at the edge ofendospore, that may be caused by the curvature/edge effectsand (iii) A stiffer area in the centre of endospore. The flat areais hypothesized to be because the protoplasm of mother cell hasbeen used to build endospore multilayers causing an increasein stiffness. Another possibility is that that the indenter may‘feel’ more of the underlying stiff substrate since the sampleis very thin at this area (Lin & Horkay, 2008). However, thisprimarily occurs when the radius of contact area is compara-ble to the sample thickness (Akhremitchev & Walker, 1999).The AFM height images show that the thickness of this areais around 120 nm, whereas the radius of tip (close to con-tact area) is only around 10 nm. Thus, the ‘finite thicknesseffect’ may be neglected for this study. The central stiff area ishypothesized to be because of endospore multilayers causingan increase in stiffness (McKenney et al., 2013). The under-lying endospore can be clearly detected at this stage. A high-resolution elasticity map obtained over the entire endosporearea (Fig. 6) further delineates the clear characteristic elas-ticity distributions. Here, we show measurements of elasticityover 2500 points (50 × 50 grid) over a smaller area of thecell surface. As expected, elasticity map on the 42 h sample(released spore, Fig. 5C) reveals a similar elasticity distribu-tion and Young’s modulus with the 24 h sample, implying thebump on the topographical image at this time period is indeed

an underlying endospore. Taken together, our nanoindenta-tion data further confirm the formation of endospore from thespatially observed progression of this process.

Finally, we investigated mature spores harvested fromsporulation medium and air dried. The high-resolution map ofthe entire spore is shown in Figure 7 in the form of an overlayof mechanical data on the surface topography. This image isobtained by the simultaneous capture of both mechanical andtopographical data in a single scan that is completed under30 min. Although the spatial resolution is therefore some-what limited in comparison to Figures 5 and 6, we wishedto demonstrate this technique as a fast method to extractinformation from the cell/spore. Surface ridges on B. cereusas observed through nanoscale imaging in earlier reportedworks are clearly seen in the topographical images (Plompet al., 2005b; Plomp & Malkin, 2009). Interestingly, theseareas are also clearly observed as softer regions of the sporesurface. This is consistent with earlier hypotheses that in anair-dried state, the decreased spore core and cortex dimen-sions result in ridges formed due to the folding of the sporecoat (Plomp et al., 2005b). The nanoindentation therefore re-veals this configuration on the raised folds. The progression ofthe average modulus across this area (�200 data points) dur-ing the process of spore formation is shown in Figure 7B. Themoduli at the 24 h and newly released spore are very similar,whereas that of the air dried spore is around 50% higher. Thisis expected owing to the process of dehydration that resultsin mechanically robust spores. The average modulus of 5.1 ±0.3 GPa is consistent with reported values of 6.6 ± 0.4 GPa onmature Aspergillus nidulans spores (Zhao et al., 2005) and B.anthracis spores (Xing et al., 2014) . Overall, the resultant in-crease in mechanical strength of the spores contributes to theirmechanical robustness and resiliency. It must be noted herethat Bacillus (endo) spores, as indeed several cellular systems,have a high curvature. Therefore, an accurate determination


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Fig. 7. (A) Nanomechanical map formed by overlay of elasticity data on the topography of a Bacillus cereus spore. The green arrows show the positionof ridges on the surface of the spore. (B) Change in elastic modulus across the centre region of the cells during the process of sporulation (�200 pointsmeasured across five samples). The elastic modulus increases nearly 300% from the vegetative cell to the mature spore.

of the exact elasticity values across the entire cell, especially atthe edges is challenging. In our results, a softer ring-like areasurrounds the stiffer centre area of endospore. This may bedue to the curvature/edge effects of nanoindentation as wellas the collapsed exosporium of the sample during air drying.However, the flat centre area of endospore provides reliableelasticity values to confirm our observations.

Summary and conclusions

The tracking of bacterial sporulation, from the vegetative cellto a mature spore, is critical for better understanding of thisfascinating natural survival of challenged or stressed Bacilluscells. In this work, we demonstrate that AFM imaging can beused to monitor the morphology and mechanical changes ofB. cereus cells during the sporulation process at the nanoscale.High-resolution images at the micro- and nanoscales clearlyshow the morphogenesis of endospore and mature endosporeover time after the sporulation was triggered. This is char-acterized by important changes on the surface of the cell it-self that are observable at the single cell level. Moreover, theuse of AFM-based elasticity mapping indicates distinct elas-ticity changes from the vegetative cell to cell with incipientendospores, as well as similarity of elasticity distribution andvalues between endospore within the cell and released matureendospore. As the earliest phases of spore formation coupledwith nanomechanically distinct cellular constructs (vegeta-tive cells, developing and mature spores) can be observed, thisapproach offers a new way to investigate Bacillus sporulationas it relates to environmental triggers, biofilm interactionsand complex microbial communities. This work therefore pro-vides a unique look into this vital natural process. As a simple,fast and versatile platform that requires hardly any samplepreparation, the AFM can be helpful for understanding howthe bacteria form endospores from both a morphological andnanomechanical perspective at the single cell or ensemblelevel.

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Supporting Information

Additional Supporting information may be found in the onlineversion of this article at the publisher’s website:

Supplementary information contains AFM images of vegeta-tive cell and spore taken under liquid, schematic of nanoin-dentation with sample curves for modulus calculation and theraw data used for Figure 5.Fig. S1. B. cereus vegetative cells and spores in liquid. (A) and(C) are height maps of vegetative cells and spore, respectively.(B) and (D) are the corresponding amplitude images.Fig. S2. Schematic of nanoindentation experiments. Exampleof the modulus data for a vegetative cell (soft) versus the stifferspore surface.Fig. S3. Unprocessed data showing the elastic modulus (toprow) for the cells in Figure 5. Bottom row is the overlay of themodulus data with the surface topography.


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Morphological and mechanical imaging of Bacillus cereus spore formation at the nanoscale