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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 2011, p. 3102–3114 Vol. 77, No. 90099-2240/11/$12.00 doi:10.1128/AEM.01262-10Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Are the Soft, Liquid-Like Structures Detected around Bacteria byAmbient Dynamic Atomic Force Microscopy Capsules?�

A. Mendez-Vilas,1,2* L. Labajos-Broncano,1,2 J. Perera-Nunez,1,2 and M. L. Gonzalez-Martín1,2*Department of Applied Physics, University of Extremadura, Avda. Elvas s/n, 06071 Badajoz, Spain,1 and

Networking Research Center on Bioengineering, Biomaterials andNanomedicine (CIBER-BBN), Badajoz, Spain2

Received 28 May 2010/Accepted 2 March 2011

High-resolution imaging of bacterial capsules by microscopy is of paramount importance in microbiologydue to their role in pathogenesis. This is, however, quite a challenging task due to their delicate nature. In thiscontext, recent reports have claimed successful exploitation of the capacity of atomic force microscopy (AFM)for imaging of extremely deformable (even liquid) surfaces under ambient conditions to detect bacterialcapsules in the form of tiny amounts of liquid-like substances around bacteria. In order to further explore thissupposed capacity of AFM, in this work, three staphylococcal strains have been scrutinized for the presence ofcapsules using such an AFM-based approach with a phosphate buffer and water as the suspending liquids.Similar results were obtained with the three strains. AFM showed the presence of liquid-like substancesidentical to those attributed to bacterial capsules in the previous literature. Extensive imaging and chemicalanalysis point out the central role of the suspending liquid (buffer) in the formation of these substances. Thephenomenon has been reproduced even by using nonliving particles, a finding that refutes the biological originof the liquid-like substances visualized around the cells. Deliquescence of major components of biologicalbuffers, such as K2HPO4, CaCl2, or HEPES, is proposed as the fundamental mechanism of the formation ofthese ultrasmall liquid-like structures. Such an origin could explain the high similarity of our results obtainedwith three very different strains and also the high similarity of these results to others reported in the literaturebased on other bacteria and suspending liquids. Finally, possible biological/biomedical implications of thepresence of these ultrasmall amounts of liquids wrapping microorganisms are discussed.

Bacterial extracellular polysaccharides often form very thin,hydrated, and soft capsules around bacteria, which are consid-ered virulence factors, contributing to their attachment andevasion of host defenses (31) and protecting them from desic-cation. For example, pneumonia-causing Streptococcus pneu-mococcal bacteria have noncapsulated and capsulated strains.Those lacking a capsule are easily destroyed by the host and donot cause disease, whereas the capsulated ones resist phago-cytosis (14). Capsules have been found both in Gram-positiveand Gram-negative bacteria, and even some fungi such asCryptococcus neoformans have been shown to express a capsule(11). Despite their extraordinary biological/biomedical impor-tance, their extremely delicate nature can make them elusive todetection/imaging by high-resolution techniques such as elec-tron microscopy, as they are poorly preserved during the de-hydration step (8). Some authors have even suggested that thecapsules might be present in the form of nanometer-thicksurface envelopes, which would also make them elusive todetection by optical microscopy or even biochemical methodssuch as India ink staining (15). For this reason, there areongoing efforts toward the direct detection of these polymericstructures under minimally perturbing conditions. AFM has

been proposed as a high (nanometer)-resolution techniquethat could provide direct evidence of the presence of bacterialcapsules under ambient conditions. Indeed, AFM has provedcapable of scanning the surface of liquids, and this is beingcurrently explored in areas such as the study of wetting prop-erties at ultrasmall scales (19). In this sense, several works haverecently reported the observation of capsules using ambientAFM (25, 27, 30, 31) in the form of tiny amounts of a liquid-like substance around bacteria. Theoretical models are beingcurrently developed for fluid microbial capsules (3). Thoseliquid-like substances would, of course, be heavily perturbed byvacuum-based techniques such as electron microscopy. Otherauthors have used the capability of AFM to measure interac-tion forces rather than as a nanoimaging tool to study extra-cellular polymers (32).

In line with those previous reports, in this work, three dif-ferent strains of Staphylococcus epidermidis differing in theability to produce extracellular polymeric substances (EPS)(10) have been scrutinized in order to detect if their capsules,if present, could be imaged by using ambient AFM. The choiceof this technique is based on the higher resolution achievedwith respect to imaging inside liquids (7). In fact, other authorshave reported limited success in imaging encapsulated bacteriain a liquid medium due the poor adhesion of cells to thesubstrate and the high level of capsular softness (25). Staphy-lococci are extremely desiccation tolerant, which enhancestheir potential for survival and transmission (2). S. epidermidisis among the most prevalent species in biomaterial-relatedinfections, as they are introduced into the body directly fromthe patient’s skin during surgical operations. Some authors

* Corresponding author. Mailing address for A. Mendez-Vilas: Net-working Research Center on Bioengineering, Biomaterials and Nano-medicine (CIBER-BBN), Badajoz, Spain. Phone: 34-924258615. Fax:34-924263053. E-mail: [email protected]. Mailing address for M.L. Gonzalez-Martín: Department of Applied Physics, University ofExtremadura, Avda. Elvas s/n, 06071 Badajoz, Spain. Phone: 34-924289532. Fax: 34-924289651. E-mail: [email protected].

� Published ahead of print on 11 March 2011.

3102

have suggested that all staphylococci are encapsulated in spiteof the difficulties in detecting the capsular material (13, 15).

MATERIALS AND METHODS

Suspending liquids and chemicals. Several suspending liquids were used inthis study with either bacteria or nonbiological particles: KPi buffer (0.87 gliter�1 K2HPO4, 0.68 g liter�1 KH2PO4, pH 7), distilled water, a 0.5 mM HEPESsolution (Sigma-Aldrich), and phosphate-buffered saline (PBS; prepared from0.01 M KPi buffer by adding 8.76 g liter�1 NaCl). Solid powders were used forthe deliquescence experiments, i.e., HEPES (99.5% titration; Sigma-Aldrich),CaCl2 (CODEX; Carlo Erba, Milan, Italy), and K2HPO4 � KH2PO4 (MERCK,Darmstadt, Germany). Inorganic alumina particles (particle size, 0.1 �m; Good-fellow, Huntingdon, United Kingdom) were used to reproduce the experimentsdone with bacteria using nonbiological particles. The particle suspension wasprepared to an optical density at 600 nm of 0.6.

Microorganisms. Three strains of S. epidermidis were tested, i.e., ATCC 12228(a non-EPS producer), ATCC 35984 (a high EPS producer), and ATCC 35983(an intermediate EPS producer) (16). Bacteria were stored at �80°C in porousbeads (Microbank; Pro-Lab Diagnostics, Austin, TX). Blood agar plates wereinoculated from the frozen stock and incubated at 37°C to obtain cultures. Thisculture was used to inoculate 75 ml of Trypticase soy broth (BBL Becton Dick-inson, Cockeysville, MD) for 24 h at 37°C. The cells were harvested by centrif-ugation for 5 min at 1,000 � g (Sorvall TC6; Dupont, Newtown, CT) and washedtwice with and finally resuspended in KPi or distilled water to an optical densityat 600 nm of 0.6. Drops of 15 �l of a bacterial suspension were transferred toclean glass slides and left to air dry, leaving a circular spot after evaporation.

AFM. A PicoLE AFM apparatus from Agilent Technologies, operated intapping mode under ambient conditions (40 to 50% relative humidity [RH]), wasused to analyze the spot that was left after evaporation of the bacterial suspen-sion droplet. Due to mechanisms explained in Results and Discussion, a quiteheterogeneous spot, displaying an increasing amount of mass (bacteria andbuffer residue) toward the spot periphery, was produced. Due to this macro-scopic heterogeneity, extensive imaging was performed in order to give spatiallyresolved and representative results for each area. A total of 626 AFM images ofbacteria were analyzed in the course of the present study. The cantilever used(NSC11/Cr-Au; MikroMasch Spain, Madrid, Spain) had a nominal spring con-stant of 3 N/m and a nominal resonance frequency of 60 kHz (as supplied by themanufacturer). In our study, we managed to obtain good (noise-free) imagesusing these k � 3 N/m cantilevers, but this is compatible with the fact that otherauthors have obtained good images using softer ones, since stability during thescanning of such delicate surfaces does not rely only on the cantilever’s stiffness.For example, Stukalov et al. (30) used softer cantilevers. The final success orfailure of the imaging experiments will rely heavily on aspects such as thewettability (its affinity for the liquid being scanned) of the tip apex, which cannotbe directly and easily measured. When we initiated this study, we chose to workwith stiffer cantilevers since their higher k would provide them with greaterkinetic energy (1/2k�x2�, where x is the vibration amplitude) to overcome thecapillary forces between it and the liquid-like structures. But as already said,there is still not any general rule for safely imaging such soft surfaces using AFM.The tips are Au covered. At least three independent experiments were per-formed for each bacterial strain. The Gwyddion AFM software has been used forthe presentation and analysis of AFM images.

X-ray photoelectron spectroscopy (XPS). The surface chemistry of the spotwas analyzed in an attempt to get some more insight into the results obtained byAFM. Data were collected at equidistant points along a diameter of the spot. Weused a K-alpha model (Thermo Scientific, Loughsborough, United Kingdom).Measurements were made using an excitation source of Al-K� radiation (1,486.6eV) under a chamber pressure of 10�9 torr (1 torr � 1.333 � 102 Pa). Aphotoelectron detector was placed at a normal position (90° takeoff angle) withregard to the sample. The X-ray beam diameter was 400 �m. Survey spectracollected for deducing the atomic concentration of the elements present in thesample were recorded with a pass energy of 100 eV. In order to detect the bufferresidue on the spot, the two most representative elements of the buffer (P and K)where mapped using the peaks corresponding to the P2p and K2p electronicorbitals. These were recorded with a pass energy of 20 eV. We performed 10 and20 scans for the survey and high-resolution spectra, respectively.

Optical microscopy. A series of experiments were accomplished in order toshow the deliquescent/nondeliquescent behavior of some of the major compo-nents of biological buffers. To this end, these components were spread over aglass support in the form of a powder and held inside a closed vessel containinga bulk water reservoir. In order to image the evolution of the powders uponwater vapor uptake, images were taken at regular intervals using a zoom stereo-

microscope (SZX10; Olympus) with a V-CMAD3 adapter (Olympus). Imageswere recorded using a CV-S3200N color charge-coupled device camera (JAI,Yokohama, Japan) connected to a personal computer.

Cell viability. In the last stage of this study, we explored if there was anyrelationship between the condition of the microorganisms as revealed by AFMand their viability. Some preliminary results of this viability study are presented.It was conducted using the commercially available LIVE/DEAD BacLight bac-terial viability kit, a two-color fluorescence assay that simultaneously determineslive (seen as green) and dead (seen as red) cell numbers.

RESULTS AND DISCUSSION

Study by AFM. Drops of bacterial suspensions in KPi weredeposited onto glass substrata and left to evaporate prior toAFM imaging. The exact structure of the bacterial spot de-pends on a variety of factors, such as the evaporation condi-tions or even irregularities of the substratum. In any case, itwas always observed that a clear peripheral ring was formed,indicating a higher concentration of mass in that area, togetherwith a clearer inner zone. Evaporating droplets carrying hy-drophobic inorganic or organic particles, including microor-ganisms, have been shown to give rise to rings (26). This phe-nomenon is usually referred as a “coffee ring” effect and wasdeeply studied by Deegan et al. (6). One of the major factorscontributing to this effect is the higher evaporation flux at thepinned contact line with respect to the center of the droplet,which produces a mass flow toward the three-phase contactline. It was evident that the spot size was larger for S. epider-midis ATCC 12228. This is likely due to the higher hydropho-bicity of this strain with respect to the other two, as suggestedby other work (20). Hydrophobic bacteria have been shown toact as surfactants (35). As such, they could have lowered thewater surface tension, thus giving rise to larger spots. In termsof the distribution of the bacterial sediment on the spot, amore homogeneous distribution was visually seen for S. epider-midis ATCC 35984 and ATCC 35983 with respect to hydro-phobic strain ATCC 12228, thus displaying a thinner ring. Thisbehavior is in accord with other work showing the redistribu-tion of aqueous solutions carrying hydrophilic or hydrophobicparticles in suspension (29).

The AFM study was conducted in a radial direction since aradial distribution of deposited mass on the glass slides wasobserved after evaporation. Extensive AFM imaging gaverather similar results for the three strains. To facilitate reading,we will refer to S. epidermidis ATCC 12228 as an EPS� strain,ATCC 35984 as an EPS�� strain, and ATCC 35983 as anEPS� strain.

While imaging the EPS� strain at the center of the spot (Fig.1a), many of the images taken at a 20- to 40-�m scale showedno bacteria there. In this part of the spot, imaging in tappingmode was straightforward and stable. When bacteria wereseen, they were usually present as aggregates with well-definedborders and showing no external substances between them, ingeneral. Only some of the isolated bacterial cells showed asmall amount of a liquid-like substance around them. A qual-itatively very similar result was obtained with the EPS�� andEPS� strains (Fig. 2a and 3a and b), with small amounts ofliquid-like substances around the bacteria when scanning at thecenter of the spot. With the EPS�� and EPS� strains, it wasmost difficult to locate the center of the spot by AFM as these

VOL. 77, 2011 LIQUID-WRAPPED BACTERIAL CELLS IMAGED BY AFM 3103

FIG. 1. AFM images of S. epidermidis ATCC 12228 cells on glass (the suspending liquid was KPi buffer). (a) Two amplitude images of thebacterial cells at the center of the spot. Note the small amount of amorphous substance that surrounds the isolated bacteria in the image on theright. In the following panels, the left side is an amplitude image and the right one is a phase image. (b) Bacterial cells detected in intermediatezones between the spot center and periphery. (c) Bacterial cells detected in peripheral areas. (d) Example of an area at the periphery showingcomplete coverage by a liquid-like substance with the glass substratum no longer visible.

3104 MENDEZ-VILAS ET AL. APPL. ENVIRON. MICROBIOL.

FIG. 2. AFM images of S. epidermidis ATCC 35984 cells on glass (the suspending liquid was KPi buffer). (a) Amplitude image of the bacterialcells at the center of the spot (b, c, and d) Amplitude (left) and phase (right) images of the bacterial cells detected toward peripheral areas.


FIG. 3. AFM amplitude (left) and phase (right) images of S. epidermidis ATCC 5983 cells on glass (the suspending liquid was KPi buffer). (a,b) At the center of the spot; (c, d) toward peripheral areas.


bacterial suspensions produced small spots. Thus, the term“center” should be taken as approximate.

While moving to peripheral areas, bacteria were always seenin all of the 20- to 40-�m images acquired. In these areas, thethree strains frequently appeared as embedded in an amor-phous liquid-like substance (Fig. 1b, 2b and c, and 3c). Theseamorphous substances displayed a strong contrast in the phasesignal with respect to the substratum and cell surface, indicat-ing different chemical or mechanical properties. Phase imagecontrast arises due to variations in energy dissipation betweenthe tip and the sample, which can be caused by adhesion orviscoelasticity (4, 12). Clearly, such a strong contrast in phaseshould be caused by the energy dissipated in the extremelydeformable liquid-like structure/air interface. Suo et al. (31)obtained very similar liquid-like structures, identifying them asbacterial capsules, while imaging the bacterium Salmonellaenterica serovar Typhimurium under ambient conditions, usinga HEPES solution as the suspending medium. In their case,phase images revealed dark-colored liquid-like substancesaround their bacteria. Stukalov et al. (30) also obtained similardark-colored structures around cells that they attributed tocapsules in AFM phase images. Obst and Dittrich (25) ob-tained liquid-like substances very similar to those observed byus at the periphery of the spots, which displayed a bright colorin phase images. In our study, most of the images revealed theliquid-like substances as brighter zones in phase images. How-ever, we found the contrast to be very sensitive to the set pointamplitude. In fact, it could be changed from bright to dark byreducing the set point amplitude, i.e., making the tappingharder. Inversion in topography and phase signals with variouslevels of tapping damping has indeed been reported for softmaterials (12, 24). Usually, such a reduction in the set pointamplitude ratio led to better images, reducing the “shadow”effect visible on some bacteria (always in the direction of scan-ning) but could also eventually favor the capture of the tip bythe liquid-like substance, thus leading to signal loss. In thosecases, the cantilever was pushed away from the sample andreengaged.

While moving to even more peripheral areas (trying to lo-cate the tip just at the ring), an increasing amount of thisliquid-like substance was observed for the three strains (Fig. 1cand d, 2d, and 3d). At these points, scanning frequently be-came extremely unstable and imaging parameters had to beiteratively controlled during the acquisition of large images soas to maintain the signal. This evidenced a very soft and/oradhesive surface. It was usually necessary to repetitively imagethe same area until a noise-free image could be obtained.Teschke reported that images could be not obtained in some ofthe areas explored in an evaporated bacterial spot due to theirhighly viscoelastic properties (33). This is compatible with ourexperience, and it is possible that they were moving on areasextensively covered by soft liquid-like substances that madetheir imaging so unstable. In many cases, coverage was soextensive than only a small fraction of the bacteria, the upperpart, was visible. For example, Fig. 1d and 3d show areas wherethe glass substratum is almost or totally not seen. While top-ographic amplitude images show these liquid-like structures tobe smooth with no specific internal structure, phase imagesfrequently revealed them to be heterogeneous, showing thepresence of both branched and continuous compact structures

(Fig. 1c or d or 2b). Although their origin is unclear, they couldbe caused by the dissolution or detachment of bacterial surfacemolecules into the liquid-like substance or by the release ofintracellular substances due to cell lysis. In any case, this isindeed a striking demonstration of the exquisite sensitivity ofphase imaging to surface heterogeneities, even in the case ofextremely soft surfaces like these. The absolute value of thedifference between the phase angle of the substratum and thatof the liquid-like substances ranged from about 5° to about 31°,with no significant differences found in the three studies donewith the three different bacteria. This is quite similar to theangles reported by Stukalov et al. (30), who found values of 15°to 35° when bacteria were exposed to HEPES buffer with micaas the substratum. Obst and Dittrich (25) did not report nu-merical values, but based on their images (gray scale in phaseimages from 0° to 12°), it is likely that their results are similarin terms of phase angles.

At this point, the first important observation is that theamount of this liquid-like substance increases as we movetoward the periphery of the spot. This kind of substance, im-aged by ambient AFM, especially when it appears aroundsingle bacteria (as in our center-intermediate zones), is virtu-ally identical to what has been associated with bacterial cap-sules in the some other investigations (30, 31). When detectedin larger quantities (as in our peripheral areas), they have beenattributed to EPS produced by the bacteria (25). The resultsobtained in this work, where extensive imaging covering dif-ferent zones of the macroscopically heterogeneous system wasperformed, even in those which are recognizably hardly scan-nable, suggest a physical or chemical origin of those sub-stances, since the amounts in which they are present just de-pend on where measurements are made in the spot. Also, theobservation that cumuli of cells were completely embedded,sharing the same “capsular material,” as well as the enormousamounts (compared to the cell size) observed in areas at theperiphery, suggested a possible nonbiological origin of thismaterial. Imaging of these liquid-like substances at the samelocation for several days did not show any appreciable changein their amount, suggesting that a rapid equilibrium wasreached once the liquid was visually seen to macroscopicallyevaporate before the beginning of the imaging experiences.This certainly rules out the possibility that these structures arecomposed solely of water, as such ultrasmall volumes wouldrapidly evaporate.

Study by XPS. To shed light on the origin of these liquid-likestructures, and specifically on the spatial variation observed, anXPS chemical analysis of the spots was performed. XPS spec-tra were recorded at equidistant points along a spot diameter.The distribution of the two most representative elements of thebuffer, P and K, was mapped. The results, presented in Fig. 4afor the EPS� strain, showed a gradual increase in the surfaceconcentration of these ions toward the periphery of the spot,thus matching the trend observed in the appearance of theliquid-like substances as shown by AFM imaging. The samebehavior was obtained in the chemical analysis of the spots forthe other two strains. This directly points to a close relation-ship between the surface concentration in buffer ions producedduring evaporation and the formation of the liquid-like sub-stances, a fact that also suggests a nonbiological origin.

KPi buffer is composed of an equimolar mixture of K2HPO4


and KH2PO4. Therefore, the relative atomic concentrationratio of K to P is 3:2. When this relative abundance was ob-tained from the XPS survey spectra, it turned out to be veryclose to that stoichiometric ratio, indicating that the detectedK and P atoms came from the buffer used and were of non-biological origin (Fig. 4b).

Taken together, the AFM and XPS data clearly revealedthat the appearance of the extracellular liquid-like substanceswas related to the local concentration of the buffer ions.

Suspension of bacteria in H2O. In order to test this hypoth-esis about the influence of buffer ions, the same AFM exper-iments were performed using distilled water instead of KPibuffer as the suspending liquid. Sessile droplets of bacterialsuspensions were also left to evaporate over the glass substra-tum. Extensive imaging was then performed by AFM in differ-ent areas of the macroscopic spot. It is noteworthy that liquid-like substances were never observed (Fig. 5). As a result ofosmotic shock, cells appeared enlarged and distorted but there

was no evidence of the presence of those substances, regardlessof the zone where imaging was done. This confirms the centralrole of buffer ions in the observation of those liquid structures.Of course, this study included images of a large variety of sizes.When extracellular substances were observed around bacteria(when using KPi buffer), they were clearly resolved in imagesof a few or a few tens of micrometers. Most of the imagesshown here are 10 to 20 �m in size. For a resolution of 512 by512 points, this gives 15,000 nm/512 points, 30 nm/point, aquantity which is comparable to the tip apex dimensions.

In summary, no evidence of extracellular liquid-like sub-stances has been found when the suspending liquid is water.Clearly, their origin must be linked to the ions present in thebuffers used.

Replacement of bacteria with nonbiological (Al2O3) parti-cles. To further clarify the origin of the liquid-like substancessurrounding bacterial cells and specifically to find out if thebacteria could play some role in their appearance, structure,

FIG. 4. Three-dimensional representation of XPS spectra collected at equidistant points along a diameter of the evaporated spot (for S.epidermidis ATCC 12228; the suspending liquid was KPi buffer) showing the variation of buffer elements P and K. Identical behavior was obtainedwith the other two strains. (b) Graph showing a match between the surface concentration of K and that of P multiplied by 3/2 along the diameterof the spot, which is the proportion of these elements in KPi buffer.


and/or stability, experiments with nonbiological particles(Al2O3 powder) were performed. As the suspending liquid weused KPi buffer, since it produced the liquid-like substancesaround our S. epidermidis cells, and a HEPES solution, as thisbuffer has been shown to result in the formation of similarliquid-like substances in other work (31).

With KPi as the suspending liquid, both isolated and aggre-gated alumina particles were found at the center of the spot,with no evidence of any kind of liquid-like substances in thisarea (Fig. 6a). When moving toward the spot periphery, abehavior very similar to that encountered while imaging bac-teria was found, imaging becoming increasingly unstable, thusrequiring trained users. Again, liquid-like substances were de-tected around the alumina particles in this area (Fig. 6b),displaying a high contrast in the phase signal. The same patternwas followed when using HEPES solution, with naked particlesat the center of the spot (Fig. 6c), and particles surrounded byincreasing amounts of liquid-like substances when moving to-ward peripheral areas (Fig. 6d).

The results obtained with these experiments performed withnonbiological particles definitively show that there is no needto invoke any biological structure or process for reproducingthe phenomenon observed for bacteria.

Mechanism of formation of the observed liquid-like sub-stances and implications. Our experiments have clearly shownthat ultrasmall amounts of liquid surrounding bacterial cellscommonly observed by AFM under ambient conditions arerelated to the buffers used. At this point, what is still unclear isthe mechanism behind this extremely evaporation-resistant be-havior of these tiny (femto- or attoliter) liquid volumes. Such

extremely small volumes of conventional liquids should evap-orate in a few seconds under ambient conditions. When wateris used as the suspending liquid, no liquid substances are seen,which is consistent with the evaporative behavior of water (forexample, a “large” water microdroplet with a volume of 10�m3 evaporates in 10 s in a 99% RH atmosphere) (1). Salts areknown to have little effect on the evaporation of water, but astabilization as strong and long lasting as that observed in thiswork cannot be explained based only on a solute effect.

We propose here deliquescence as the basic chemical prop-erty responsible of the appearance of the observed liquid-likesubstances. Deliquescence is a phenomenon by which certainsubstances are able to absorb moisture from the ambient airand dissolve in it (17). It should not be confused with hygro-scopicity, by which these substances adsorb or absorb moisturebut do not form a solution upon absorption. Many of the saltsused in common biological buffers are deliquescent. In the caseof KPi, one of its components, K2HPO4, is highly deliquescentwhile the other one, KH2PO4, is not. CaCl2, commonly used inbiological buffers, is among the most deliquescent salts, jointlywith other divalent chlorides such as MgCl2 or ZnCl2. It is,indeed, commercially available as an air dehumidifier. In thecase of HEPES, no information seems to be available, al-though its piperazine moiety is known to be deliquescent(DrugBank primary accession number DB00592).

To demonstrate the phenomenon, optical micrographs weretaken inside a closed vessel containing powders of the sub-stances studied deposited over a glass support and a bulk waterreservoir. As can be seen in the photographic sequence in Fig.7, CaCl2, K2HPO4, and HEPES became completely liquid

FIG. 5. AFM amplitude images of S. epidermidis ATCC 12228 (a) and ATCC 35984 (b) cells on glass (the suspending liquid was water). Thesame result was obtained with S. epidermidis ATCC 35983, with no liquid-like substances observed at any place on the sample.


FIG. 6. Evaporated spot from a KPi buffer solution (a, b) and a HEPES solution (c, d) containing nonliving particles (alumina powder). (a, c)Cumuli of naked alumina particles are visible at the center of the spot. (b, d) In peripheral areas, alumina particles are surrounded by a liquid-likesubstance which is virtually identical to those detected in the case of bacterial suspensions. Both amplitude (left) and phase (right) images areshown.


upon contact solely with water vapor. Clearly, KH2PO4, theother KPi component, is not deliquescent and images showedno temporal evolution observed to up to 5 h (images becamemore and more blurry because of water condensation at thetop of the vessel).

Deliquescence at the micro- or nanometer scale could thenreadily explain why the liquid-like substances appear aroundbacterial cells, as was observed by us. On the other hand, theaqueous nature (water plus deliquescent salts) of the observedsubstances is compatible with the low contact angle they ex-hibit with respect to the hydrophilic glass support (20° 7°)(Fig. 8). Low contact angles were also obtained in the experi-

ments using alumina powder, with average values of 25° 7°and 22° 6° when the suspending liquids were KPi andHEPES buffer, respectively. This proposed mechanism couldalso explain the results obtained by Suo et al. (31) using bufferscontaining deliquescent components (HEPES and CaCl2) andcould justify the high similarity of the Raman spectra obtainedfor solid HEPES and for the capsule in that report. Also, sucha physicochemical (not biological) origin could explain whythese authors obtained very similar results for a Gram-negative(Escherichia coli) and a Gram-positive (S. Typhimurium) bac-terium. The use of other biological buffers such as N,N-bis(2-hydroxyethyl)piperazine (BHEP) or morpholinepropanesul-fonic acid (MOPS) also resulted in the observation of“bacterial capsules,” but they are very similar to HEPES. Inthe case of PBS, these authors did not observe the capsules.This is consistent with our deliquescence-based explanation, asthe major component of PBS, NaCl, is deliquescent only athigh humidity (75 to 76%) (34), so it is possible that theirambient humidity did not reach that critical value. Also, al-though PBS components such as K2HPO4 are deliquescent,they are only a minor component, and in addition, the largeNaCl crystals produced could easily interfere with the obser-vation of the liquid-like substances. Indeed, in performing ex-tensive AFM imaging, we were able to detect stable liquidsubstances surrounding crystals formed upon evaporation ofPBS (as exemplified in Fig. 9). However, this proved to be verytime-consuming since the large dimensions of the NaCl crystalsfrequently exceeded the z range of the AFM scanner, thusmaking imaging quite unstable.

The sensitivity of the deliquescence phenomenon to the

FIG. 7. Evolution of powders of different substances used in biological buffers when exposed to a humid atmosphere.

FIG. 8. Example of a topographical profile of the liquid-like sub-stance surrounding a bacterial cell, where the contact angle formedwith the glass substratum can be measured (schematically drawn in theinset).


ambient humidity will result in differences in the amounts ofliquid-like substances detected, depending on the specific en-vironmental conditions of the facility where experiments arerun. While it is likely that no liquid-like substances will beobtained in facilities established in very dry environments, theopposite will likely happen in labs situated in very humid en-vironments. Under low-humidity conditions, evaporation ofbuffer residue will proceed until equilibrium is reached andprogressive solidification might occur. This could explain theprogressive hardening of the liquid-like substances (supposedto be bacterial capsules) observed by Suo et al. (31). Ourproposed mechanism could also explain why Stukalov et al.(30) succeeded in observing capsules in some strains whenusing AFM but not when using transmission electron micros-copy (TEM). Indeed, those results suggested that AFM ismore reliable than TEM, as AFM unambiguously showed thepresence capsules. Our results show that this might not be thecase because these “capsules” detected by AFM can be formedjust by water uptake by deliquescent buffer components andthat the reason for their invisibility to TEM is liquid evapora-tion under the vacuum imposed by this technique. We notethat this phenomenon will likely also be visible when the bufferis applied to the substratum in the form of a film instead of adroplet, since liquid films ultimately break into droplets atadvanced stages of evaporation. Each one of these dropletscarries its own contact line, with the potential of concentratingthe ions at it to a value high enough to collect enough waterfrom the ambient to give rise to measurable liquid structures.Of course, at a certain buffer concentration, the larger thedroplet, the higher the quantity of buffer ions and the higherthe amount of liquid-like substances that will appear. Theamount of liquid-like substances will be proportional to theconcentration of the buffer used.

High-resolution imaging of microorganisms in gaseous en-vironments can be of interest in a largely diverse set of fields,such as microbial ultrastructure with nanometer resolution orthe study of mechanisms of microbial tolerance to stress con-

ditions, such as desiccation, which is important in food science(9, 21, 22). However, it has to be emphasized that even thoughAFM does not impose conditions as aggressive as those ofelectron microscopy, its results can be strongly affected byother subtle factors, as demonstrated in this work. Also, thiswork emphasizes that the sessile-droplet (drop-and-dry)method, which is very common in some biological assays, givesrise to an extremely heterogeneous system, as a result of phys-ical flows which take place during the evaporation of sessiledroplets. For this reason, performing extensive imaging is aprerequisite when trying to characterize such a macroscopi-cally heterogeneous system with a local technique such asAFM. This evaporation-induced heterogeneity is indeed a rec-ognized problem in other areas involving droplets contactingsolids, such as in the fabrication of microarrays, whose reliabil-ity can be seriously compromised by the heterogeneous distri-bution produced (18). A negative effect of this phenomenonhas also been suggested in DNA hybridization studies usingsessile droplets, in which most of the DNA can be lost at thespot periphery, potentially giving false-negative results (28).

An interesting implication of this work is that a sample maynot be as dry as it might look when it has been in contact withliquids containing deliquescent components and that ultra-small amounts of aqueous liquids can persist at the surface.Detection by optical microscopy could be hampered by reso-lution and transparency issues. They could also be overlookedin electron microscopy studies, as all liquid substances wouldevaporate under the vacuum imposed. As mentioned above,many extremely deliquescent salts are usually used in the for-mulation of biological buffers and culture media. When deli-quescent components are used in disinfectants or biocides ingeneral, for example, it could be interesting to explore if thesomehow wet residue which is being left might have somebiological/biomedical relevance. For example, they could pro-vide a site where surviving microorganisms could develop re-sistance or where other external microorganisms such as fungicould proliferate. In the current context of the emergence of

FIG. 9. AFM amplitude (left) and phase (right) images of a microscopic liquid-like film detected upon evaporation of a macroscopic PBSdroplet.


microbial resistance not only to antibiotics but also to chemicalagents (biocides), it is generally accepted that the time ofaction must be as short as possible, leaving no residue, as thiscould provide microenvironments for resistance development(23).

We are currently exploring some biological/biomedical con-sequences of these kinds of liquid-like substances which mightbe present under ambient conditions when deliquescent com-ponents are used in liquid formulations. This has been moti-vated by some preliminary results we have recently obtainedand some of which are presented here. In Fig. 10, we showfluorescence microscopy images corresponding to a LIVE/DEAD viability essay performed over the EPS� strain at the

center of the spot (where the bacteria are not “protected” or“stressed” by the liquid-like environment) and at the spot pe-riphery (extensively covered by the liquid-like substance), after12 h of air drying. Notably, most of the cells at the peripherywere still viable after this time (they appear green in the flu-orescence test) while most of those at the center of the spotseemed to have died (red). These preliminary results havemotivated a dedicated work to investigate the potential sensi-tivity of microorganisms of biomedical relevance to such ul-trasmall amounts of liquids. Results might be interesting in anysituation where microorganisms are in contact with liquidsundergoing evaporation, such as in disinfection procedures(e.g., hospitals), when deliquescent substances are included inthe formulation of disinfectants. As mentioned above, manydivalent chlorides are deliquescent, and chlorine, which is usu-ally incorporated in solutions in the form of chlorides, is awell-known disinfectant/antiseptic. In another field, it has in-deed been recently discovered that certain microorganisms aretaking advantage of the deliquescent nature of some mineralspresent in the Atacama Desert, the driest place on Earth, tosurvive in such a hostile environment (5).

Finally, the use of deliquescent salts to stabilize and studyultrasmall liquid droplets on solid surfaces under normal am-bient conditions, which is not possible with conventional liq-uids such as water or organic liquids due to almost instanta-neous evaporation, could provide new avenues for the study ofwetting properties at ultrasmall scales, a recently reviewed hotarea in surface science (19), since liquid topography can beaccurately tracked at high resolution using AFM.

Conclusions. Bacterial cells imaged under ambient condi-tions after exposure to a phosphate buffer appear to bewrapped in a liquid-like substance that proves to be stableunder ambient conditions and that can be successfully imagedusing tapping mode AFM. Several past investigations associ-ated these kinds of liquid-like substances with bacterial cap-sules, which are key players in microbial pathogenesis. In thiswork, by studying the occurrence of this liquid-like substancein the presence of a gradient in the surface concentration ofthe rinsing buffer ions, a correlation was found between theamount of liquid-like substances and the concentration of buf-fer ions, suggesting a physicochemical (nonbiological) origin ofthose substances. This is confirmed by the fact that the phe-nomenon is fully reproduced using nonbiological particles in-stead of bacteria. Deliquescence of major buffer components isproposed as the basic mechanism for the formation of theseultrasmall liquid volumes resisting evaporation under ambientconditions. Our results seem to rule out any biological origin ofthese extracellular structures detected. The conclusionsreached in this work were achieved with S. epidermidis cells,and thus, they might not be applicable to the other worksmentioned that also employed ambient AFM. However, sinceour images are virtually identical to those shown in such works,our proposed (simpler, nonbiological) mechanisms could atleast provide an alternative explanation for the occurrence ofsuch bacterial structures when imaged under ambient condi-tions.

ACKNOWLEDGMENTS

Funding of this research has been provided by the Spanish Ministryfor Science and Technology through project MAT2009-14695-C04-01.

FIG. 10. Fluorescence microscopy images corresponding to LIVE/DEAD viability tests (S. epidermidis ATCC 12228; the suspendingliquid was KPi buffer) performed along a diameter of the spot after12 h of air drying. (a, c) At two diametrically opposed points on theperiphery. (b) At the center.


L.L.-B. thanks Junta de Extremadura and FEDER for financial sup-port under project PRI08A141. M.L.G.-M. acknowledges Junta deExtremadura and FEDER for an I3 Fellowship. J.P.-N. acknowledgesJunta de Extremadura for a predoctoral grant.

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