The response of dual-species bacterial biofilm to 2% and 5% NaOCl mixed with etidronic acid: A laboratory real-time evaluation using optical coherence tomography.

AIM: The addition of etidronic acid (HEDP) to sodium hypochlorite (NaOCl) could increase the antibiofilm potency of the irrigant, whilst maintaining the benefits of continuous chelation. Studies conducted so far have shown that mixing HEDP with NaOCl solutions of relatively low concentration does not compromise the antibiofilm efficacy of the irrigant. However, the working lifespan of NaOCl may decrease resulting in a reduction of its antibiofilm efficacy over time (efficiency). In this regard, continuous irrigant replenishment needs to be examined. This study investigated the response of a dual-species biofilm when challenged with 2% and 5% NaOCl mixed with HEDP for a prolonged timespan and under steady laminar flow. METHODOLOGY: Dual-species biofilms comprised of Streptococcus oralis J22 and Actinomyces naeslundii T14V-J1 were grown on human dentine discs in a constant depth film fermenter (CDFF) for 96 h. Biofilms were treated with 2% and 5% NaOCl, alone or mixed with HEDP. Irrigants were applied under steady laminar flow for 8 min. Biofilm response was evaluated by means of optical coherence tomography (OCT). Biofilm removal, biofilm disruption, rate of biofilm loss and disruption as well as bubble formation were assessed. One-way anova, Wilcoxon's signed-rank test and Kruskal-Wallis H test were performed for statistical analysis of the data. The level of significance was set at a ≤.05. RESULTS: Increasing NaOCl concentration resulted in increased biofilm removal and disruption, higher rate of biofilm loss and disruption and increased bubble formation. Mixing HEDP with NaOCl caused a delay in the antibiofilm action of the latter, without compromising its antibiofilm efficacy. CONCLUSIONS: NaOCl concentration dictates the biofilm response irrespective of the presence of HEDP. The addition of HEDP resulted in a delay in the antibiofilm action of NaOCl. This delay affects the efficiency, but not the efficacy of the irrigant over time.

The Effect of the Ultrasonic Irrigant Activation Protocol on the Removal of a Dual-species Biofilm from Artificial Lateral Canals.

INTRODUCTION: Lateral canals are particularly challenging to clean and disinfect. The aim of this study was to compare the removal efficacy of a dual-species biofilm from a lateral canal model by different ultrasonic irrigant activation protocols in vitro. METHODS: Artificial root canal models with 270 simulated lateral canals were made of polydimethylsiloxane. A dual-species biofilm (Streptococcus oralis and Actinomyces naeslundi) was grown in vitro in the lateral canals using a constant depth film fermenter. Two percent NaOCl or demineralized water was delivered by a syringe and an open-ended needle for 30 seconds and subsequently activated by an ultrasonic file for a total activation time of 30, 60, or 90 seconds divided in 1 or 3 consecutive activation cycles. In the control groups, the irrigant was allowed to rest for 30, 60, or 90 seconds. The volume of the biofilm in the lateral canal was evaluated before and after the final irrigation protocol by optical coherence tomography. The results were analyzed by 3-way factorial analysis of variance (α = 0.05). RESULTS: Irrigation with NaOCl rather than demineralized water resulted in more effective biofilm removal from the lateral canal (P < .001). Three cycles of intermittent ultrasonic activation were significantly more effective than no activation (P = .029). The total irrigant contact time did not affect biofilm removal (P = .403). CONCLUSIONS: The type of the irrigant and the ultrasonic activation protocol affected biofilm removal from artificial lateral canals. None of the compared protocols was able to eradicate the biofilm.

Secreted products of oral bacteria and biofilms impede mineralization of apical papilla stem cells in TLR-, species-, and culture-dependent fashion.

Regenerative endodontics exploits the mineralization potential of stem cells from the apical papilla (SCAPs) in order to promote root maturation of permanent immature teeth. SCAPs may encounter post-disinfection residual bacteria either in planktonic or in biofilm growth mode. Bacterial components bind to Toll-like receptors (TLRs) and trigger pro-inflammatory responses. We hypothesized that biofilm-triggered TLR activation affects the mineralization potential of human SCAPs. SCAPs were challenged with conditioned media derived from standardized dual-species biofilms and planktonic bacterial cultures and their inflammatory status and mineralization capacity were studied. Bacterial products from both growth modes (planktonic vs. biofilm) compromised cell viability, proliferation and mineralization capacity of SCAPs, but in a species- and growth mode-dependent fashion. While TLR4 expression remained unaffected, TLR2 expression was upregulated coinciding with a pro-inflammatory activation of SCAPs. Moreover, TLR and its downstream TGF-ß-associated kinase (TAK1) appeared to be blocking mineralization, as inhibition of these factors restored it. In conclusion, bacterial products promoted the pro-inflammatory status and inhibited mineralization of human SCAPs in a TLR-, species-, and culture-dependent fashion. TLR2 emerged as the pivotal mediator of these responses and further research is warranted towards the judicious manipulation of SCAPs in order to modify the untoward events of TLR-priming and signaling.

Influence of antibiotic pressure on bacterial bioluminescence, with emphasis on Staphylococcus aureus.

Bioluminescence imaging is used for longitudinal evaluation of bacteria in live animals. Clear relations exist between bacterial numbers and their bioluminescence. However, bioluminescence images of Staphylococcus aureus Xen29, S. aureus Xen36 and Escherichia coli Xen14 grown on tryptone soy agar in Etests demonstrated increased bioluminescence at sub-MICs of different antibiotics. This study aimed to further evaluate the influence of antibiotic pressure on bioluminescence in S. aureus Xen29. Bioluminescence of S. aureus Xen29, grown planktonically in tryptone soy broth, was quantified in the absence and presence of different concentrations of vancomycin, ciprofloxacin, erythromycin or chloramphenicol and was related to expression of the luxA gene under antibiotic pressure measured using real-time PCR. In the absence of antibiotics, staphylococcal bioluminescence increased over time until a maximum after ca. 6h of growth, and subsequently decreased to the detection threshold after 24h of growth owing to reduced bacterial metabolic activity. Up to MICs of the antibiotics, bioluminescence increased according to a similar pattern up to 6h of growth, but after 24h bioluminescence was higher than in the absence of antibiotics. Contrary to expectations, bioluminescence per organism (CFU) after different growth periods in the absence and at MICs of different antibiotics decreased with increasing expression of luxA. Summarising, antibiotic pressure impacts the relation between CFU and bioluminescence. Under antibiotic pressure, bioluminescence is not controlled by luxA expression but by co-factors impacting the bacterial metabolic activity. This conclusion is of utmost importance when evaluating antibiotic efficacy in live animals using bioluminescent bacterial strains.

On-demand antimicrobial release from a temperature-sensitive polymer - comparison with ad libitum release from central venous catheters.

Antimicrobial releasing biomaterial coatings have found application for instance in the fixation of orthopedic joint prostheses and central venous catheters. Most frequently, the release kinetics is such that antimicrobially-effective concentrations are only reached within the first days to weeks after implantation, leaving no local antimicrobial release available when a biomaterial-associated infection occurs later. Here we compare the ad libitum release of chlorhexidine and silver-sulfadiazine from a central venous catheter with their release from a new, on-demand release coating consisting of a temperature-sensitive copolymer of styrene and n-butyl (meth)acrylate. The copolymer can be loaded with an antimicrobial, which is released when the temperature is raised above its glass transition temperature. Ad libitum release of chlorhexidine and silver-sulfadiazine from a commercially-purchased catheter and associated antimicrobial efficacy against Staphylococcus aureus was limited to 16days. Consecutive temperature-triggers of our on-demand coating yielded little or no antimicrobial efficacy of silver-acetate release, but antimicrobially-effective chlorhexidine concentrations were observed over a time period of 60-80days. This attests to the clear advantage of on-demand coatings above ad libitum releasing coatings, that may have released their antimicrobial content before it is actually needed. Importantly, glass transition temperature of chlorhexidine loaded copolymers was lower (48°C) than of silver loaded ones (61°C), facilitating their clinical use.

Persistence of a bioluminescent Staphylococcus aureus strain on and around degradable and non-degradable surgical meshes in a murine model.

Biomaterials are increasingly used for the restoration of human function, but can become infected as a result of peri- or early post-operative bacterial contamination, although biomaterial-associated infections (BAIs) can also initiate at any time from hematogenous spreading of bacteria from an infection elsewhere in the body. Infecting bacteria in BAIs not only seek shelter in their own protective biofilm matrix, but also hide in surrounding tissue. This study compares staphylococcal persistence on and around a degradable and non-degradable surgical mesh through the use of longitudinal bioluminescence imaging in a murine model, including histological evaluation of surrounding tissue after sacrifice. Surgical meshes were first contaminated with bioluminescent Staphylococcus aureus Xen29 and subsequently subcutaneously implanted in mice. Bioluminescent staphylococci persisted on and around non-degradable meshes during the 28-day course of the study, whereas bioluminescence returned to control levels and bacteria disappeared from surrounding tissues once a degradable mesh had fully dissolved. Thus the application of degradable biomaterials yields major advantages with respect to the prevention of BAIs, as dissolution of the implant not only is associated with elimination of the protective biofilm mode of growth of the infecting organisms, but also allows the immune system to clear the surrounding tissue from infecting organisms.

A gentamicin-releasing coating for cementless hip prostheses-Longitudinal evaluation of efficacy using in vitro bio-optical imaging and its wide-spectrum antibacterial efficacy.

Cementless prostheses are increasingly popular in total hip arthroplasties. Therewith, common prophylactic measures to reduce the risk of postoperative infection like the use of antibiotic-loaded bone cements, will no longer be available. Alternative prophylactic measures may include the use of antibiotic-releasing coatings. Previously, we developed a gentamicin-releasing coating for cementless titanium hip prostheses and derived an appropriate dosing of this coating by adjusting the amount of gentamicin in the coating to match the antibacterial efficacy of clinically employed gentamicin-loaded bone cement. In this manuscript, we investigated two important issues regarding the prophylactic use of this 1 mg cm(-2) bioactive gentamicin-releasing coating in cementless total hip arthroplasty: (1) its ability to prevent bacterial growth in a geometrically relevant set-up and (2) its antibacterial spectrum. A geometrically relevant set-up was developed in which miniature titanium stems were surrounded by agar, contaminated with bioluminescent Staphylococcus aureus. Novel, bio-optical imaging was performed allowing noninvasive, longitudinal monitoring of staphylococcal growth around miniature stems with and without the gentamicin-releasing coating. Furthermore, the antibacterial efficacy of the gentamicin-releasing coating was determined against a wide variety of clinical isolates, including bioluminescent Staphylococcus aureus strains, using traditional zone of inhibition measurements. The gentamicin-releasing coating demonstrated a wide-spectrum of antibacterial efficacy and successfully prevented growth of bioluminescent staphylococci around a miniature stem mounted in bacterially contaminated agar for at least 60 h. This implies that the gentamicin-releasing coating has potential to contribute to the improvement of infection prophylaxis in cementless total hip arthroplasty.

Antibacterial efficacy of a new gentamicin-coating for cementless prostheses compared to gentamicin-loaded bone cement.

Cementless prostheses are increasingly popular but require alternative prophylactic measures than the use of antibiotic-loaded bone cements. Here, we determine the 24-h growth inhibition of gentamicin-releasing coatings from grit-blasted and porous-coated titanium alloys, and compare their antibacterial efficacies and gentamicin release-profiles to those of a commercially available gentamicin-loaded bone cement. Antibacterial efficacy increased with increasing doses of gentamicin in the coating and loading with 1.0 and 0.1 mg gentamicin/cm(2) on both grit-blasted and porous-coated samples yielded comparable efficacy to gentamicin-loaded bone cement. The coating had a higher burst release than bone cement, and also inhibited growth of gentamicin-resistant strains. Antibacterial efficacy of the gentamicin coatings disappeared after 4 days, while gentamicin-loaded bone cement exhibited efficacy over at least 7 days. Shut-down after 4 days of gentamicin-release from coatings is advantageous over the low-dosage tail-release from bone cements, as it minimizing risk of inducing antibiotic-resistant strains. Both gentamicin-loaded cement discs and gentamicin-coated titanium coupons were able to kill gentamicin-sensitive and -resistant bacteria in a simulated prothesis-related interfacial gap. In conclusion, the gentamicin coating provided similar antibacterial properties to those seen by gentamicin-loaded bone cement, implying protection of a prosthesis from being colonized by peri-operatively introduced bacteria in cementless total joint arthroplasty.

The potential for bio-optical imaging of biomaterial-associated infection in vivo.

This review presents the current state of Bioluminescence and Fluorescent Imaging technologies (BLI and FLI) as applied to Biomaterial-Associated Infections (BAI). BLI offers the opportunity to observe the in vivo course of BAI in small animals without the need to sacrifice animals at different time points after the onset of infection. BLI is highly dependent on the bacterial cell metabolism which makes BLI a strong reporter of viable bacterial presence. Fluorescent sources are generally more stable than bioluminescent ones and specifically targeted, which renders the combination of BLI and FLI a promising tool for imaging BAI. The sensitivity and spatial resolution of both imaging tools are, however, dependent on the imaging system used and the tissue characteristics, which makes the interpretation of images, in terms of the location and shape of the illuminating source, difficult. Tomographic reconstruction of the luminescent source is possible in the most modern instruments, enabling exact localization of a colonized implant material, spreading of infecting organisms in surrounding tissue and immunological tissue reactions. BLI studies on BAI have successfully distinguished between different biomaterials with respect to the development and clearance of BAI in vivo, simultaneously reducing animal use and experimental variation. It is anticipated that bio-optical imaging will become an indispensable technology for the in vivo evaluation of antimicrobial coatings.

Influence of a chitosan on oral bacterial adhesion and growth in vitro.

Generally, mechanical plaque control without chemical support is insufficient to prevent oral diseases, and an ongoing quest exists for new antimicrobials for use in oral healthcare. Chitosans are polycationic, naturally occurring antimicrobials that are rapidly finding their way into oral healthcare. In this study we determined the effects of pellicle treatment with chitosan on bacterial adhesion and growth. Chitosan caused a reduction in bacterial adhesion and was responsible for bacterial death upon contact compared with a buffer control. Exposure of adhering bacteria to a chitosan solution or a buffer control did not cause detachment, but the chitosan solution left a small proportion of the adhering bacteria alive. Growth after exposure to chitosan was similar to (Streptococcus mutans ATCC700610, Streptococcus oralis HM1, Streptococcus sobrinus HG1025) or less than (S. mutans NS, Actinomyces naeslundii HM1) that of the control, while biofilm viability after chitosan treatment was lower than that of the control, except for S. oralis HM1. Therefore, chitosan is a promising antimicrobial for use in oral healthcare.

Streptococcus mutans and Streptococcus intermedius adhesion to fibronectin films are oppositely influenced by ionic strength.

Bacterial adhesion to protein-coated surfaces is mediated by an interplay of specific and nonspecific interactions. Although nonspecific interactions are ubiquitously present, little is known about the physicochemical mechanisms of specific interactions. The aim of this paper is to determine the influence of ionic strength on the adhesion of two streptococcal strains to fibronectin films. Streptococcus mutans LT11 and Streptococcus intermedius NCTC11324 both possess antigen I/II with the ability to bind fibronectin from solution, but S. intermedius binds approximately 20x less fibronectin than does the S. mutans strain under identical conditions. Both strains as well as fibronectin films are negatively charged in low ionic strength phosphate buffered saline (PBS, 10x diluted), but bacteria appear uncharged in high ionic strength PBS. Physicochemical modeling on the basis of overall cell surface properties (cell surface hydrophobicity and zeta potentials) demonstrates that both strains should favor adhesion to fibronectin films in a high ionic strength environment as compared to in a low ionic strength environment, where electrostatic repulsion between equally charged surfaces is dominant. Adhesion of S. intermedius to fibronectin films in a parallel plate flow chamber was completely in line with this modeling, while in addition atomic force microscopy (AFM) indicated stronger adhesion forces upon retraction between fibronectin-coated tips and the cell surfaces in high ionic strength PBS than in low ionic strength PBS. Thus, the dependence of the interaction on ionic strength is dominated by the overall negative charge on the interacting surfaces. Adhesion of S. mutans to fibronectin films, however, was completely at odds with theoretical modeling, and the strain adhered best in low ionic strength PBS. Moreover, AFM indicated weaker repulsive forces upon approach between fibronectin-coated tips and the cell surfaces in low ionic strength PBS than in high ionic strength PBS. This indicated that the dependence of the interaction on ionic strength is dominated by electrostatic attraction between oppositely charged, localized domains on the interacting surfaces, despite their overall negative charge. In summary, this study shows that physicochemical modeling of bacterial adhesion to protein-coated surfaces is only valid provided the number of specific interaction sites on the cell surfaces is low, such as on S. intermedius NCTC11324. Nonspecific interactions are dominated by specific interactions if the number of specific interaction sites is large, such as on S. mutans LT11. Its ionic strength dependence indicates that the specific interaction is electrostatic in nature and operative between oppositely charged domains on the interacting surfaces, despite the generally overall negatively charged character of the surfaces.

Interaction forces between waterborne bacteria and activated carbon particles.

Activated carbons remove waterborne bacteria from potable water systems through attractive Lifshitz-van der Waals forces despite electrostatic repulsion between negatively charged cells and carbon surfaces. In this paper we quantify the interaction forces between bacteria with negatively and positively charged, mesoporous wood-based carbons, as well as with a microporous coconut carbon. To this end, we glued carbon particles to the cantilever of an atomic force microscope and measured the interaction forces upon approach and retraction of thus made tips. Waterborne Raoultella terrigena and Escherichia coli adhered weakly (1-2 nN) to different activated carbon particles, and the main difference between the activated carbons was the percentage of curves with attractive sites revealed upon traversing of a carbon particle through the bacterial EPS layer. The percentage of curves showing adhesion forces upon retraction varied between 21% and 69%, and was highest for R. terrigena with positively charged carbon (66%) and a coconut carbon (69%). Macroscopic bacterial removal by the mesoporous carbon particles increased with increasing percentages of attractive sites revealed upon traversing a carbon particle through the outer bacterial surface layer.

Interaction forces between salivary proteins and Streptococcus mutans with and without antigen I/II.

The antigen I/II family of surface proteins is expressed by oral streptococci, including Streptococcus mutans, and mediates specific binding to, among others, salivary films. The aim of this study was to investigate the interaction forces between salivary proteins and S. mutans with (LT11) and without (IB03987) antigen I/II through atomic force microscopy (AFM) and to relate these interaction forces with the adhesion of the strains to saliva-coated glass in a parallel plate flow chamber. Upon approach of the bacteria toward a saliva-coated AFM tip, both strains experienced a similar repulsive force that was significantly smaller at pH 6.8 (median 3.0 and 3.1 nN for LT11 and IB03987, respectively) than at pH 5.8 (median 4.6 and 4.7 nN). The decay length of these repulsive forces was between 19 and 37 nm. Upon retraction at pH 6.8, the combined specific and nonspecific adhesion forces were significantly stronger for the parent strain LT11 (median -0.4 nN) than for the mutant strain IB03987 (median 0.0 nN), whereas at pH 5.8 the median of the adhesion forces measured was 0.0 nN for both strains. Moreover, at pH 6.8, the parent strain LT11 adhered in significantly higher numbers (9.6 x 106 cm-2) to a salivary coating than the mutant strain IB03987 (2.5 x 106 cm-2). Similar to the difference in adhesion forces between both strains at pH 5.8, the difference in adhesion between both strains also disappeared at pH 5.8, which suggests the involvement of attractive electrostatic forces in the interaction between antigen I/II and salivary coatings. In summary, this study shows that antigen I/II at the surface of S. mutans LT11 is responsible for its increased adhesion to salivary coatings under flow through an additional attractive electrostatic force.

Chitosan adsorption to salivary pellicles.

The salivary pellicle is a negatively charged protein film, to which oral bacteria readily adhere. Chitosans are cationic biomolecules with known antimicrobial properties that can be modified in different ways to enhance its antimicrobial activity. Here, we determined the changes in surface chemical composition using X-ray photoelectron spectroscopy (XPS), in hydrophobicity by analyzing water contact angles, in charge through measuring streaming potentials, and evaluated morphology using atomic force microscopy (AFM), of salivary pellicles upon adsorption of different chitosans. The adsorption of chitosans to pellicles was chemically evident from altered carbon functionalities and the presence of an N(1s) peak at 401.1 eV as a result of protonated amines in XPS. Chitosan adsorption made the pellicle (zeta potential of untreated pellicles 29 mV) positively charged and more hydrophobic. A chemically modified chitosan (CL) and an unmodified chitosan (UC) caused aggregation of adsorbed salivary proteins, and AFM revealed clumps of protein after treatment with these chitosans, yielding an increase in pellicle surface roughness from 5.1 nm to between 16.3 and 35.6 nm for CL and UC, respectively. In summary, chitosans have a clear tendency to adsorb to salivary pellicles with a profound effect on the surface properties of the pellicle. Therefore, chitosans may provide anchoring molecules to affix antimicrobials to pellicle surfaces.

Intermolecular forces and enthalpies in the adhesion of Streptococcus mutans and an antigen I/II-deficient mutant to laminin films.

The antigen I/II family of surface proteins is expressed by most oral streptococci, including Streptococcus mutans, and mediates specific adhesion to, among other things, salivary films and extracellular matrix proteins. In this study we showed that antigen I/II-deficient S. mutans isogenic mutant IB03987 was nearly unable to adhere to laminin films under flow conditions due to a lack of specific interactions (0.8 x 10(6) and 1.1 x 10(6) cells cm(-2) at pH 5.8 and 6.8, respectively) compared with parent strain LT11 (21.8 x 10(6) and 26.1 x 10(6) cells cm(-2)). The adhesion of both the parent and mutant strains was slightly greater at pH 6.8 than at pH 5.8. In addition, atomic force microscopy (AFM) experiments demonstrated that the parent strain experienced less repulsion when it approached a laminin film than the mutant experienced. Upon retraction, combined specific and nonspecific adhesion forces were stronger for the parent strain (up to -5.0 and -4.9 nN at pH 5.8 and 6.8, respectively) than for the mutant (up to -1.5 and -2.1 nN), which was able to interact only through nonspecific interactions. Enthalpy was released upon adsorption of laminin to the surface of the parent strain but not upon adsorption of laminin to the surface of IB03987. A comparison of the adhesion forces in AFM with the adhesion forces reported for specific ligand-receptor complexes resulted in the conclusion that the number of antigen I/II binding sites for laminin on S. mutans LT11 is on the order of 6 x 10(4) sites per organism and that the sites are probably arranged along exterior surface structures, as visualized here by immunoelectron microscopy.

Removal of two waterborne pathogenic bacterial strains by activated carbon particles prior to and after charge modification.

Waterborne diseases constitute a threat to public health despite costly treatment measures aimed at removing pathogenic microorganisms from potable water supplies. This paper compared the removal of Raoultella terrigena ATCC 33257 and Escherichia coli ATCC 25922 by negatively and positively charged types of activated carbon particles. Both strains display bimodal negative zeta-potential distributions in stabilized water. Carbon particles were suspended to an equivalent external geometric surface area of 700 cm2 in 250 mL of a bacterial suspension, with shaking. Samples were taken after different durations for plate counting. Initial removal rates were less elevated for the positively charged carbon particle than expected, yielding the conclusion that bacterial adhesion under shaking is mass-transport limited. After 360 min, however, the log-reduction of the more negatively charged R. terrigena in suspension was largest for the positively charged carbon particles as compared with the negatively charged ones, although conditioning in ultrapure or tap water of positively charged carbon particles for 21 days eliminated the favorable effect of the positive charge due to counterion adsorption from the water. Removal of the less negatively charged E. coli was less affected by aging of the (positively charged) carbon particles, confirming the role of electrostatic interactions in bacterial removal by activated carbon particles. The microporous, negatively charged coconut carbon performed less than the mesoporous, positively charged carbon particle prior to conditioning but did not suffer from loss of effect after conditioning in ultrapure or tap water.

Comparison of atomic force microscopy interaction forces between bacteria and silicon nitride substrata for three commonly used immobilization methods.

Atomic force microscopy (AFM) has emerged as a powerful technique for mapping the surface morphology of biological specimens, including bacterial cells. Besides creating topographic images, AFM enables us to probe both physicochemical and mechanical properties of bacterial cell surfaces on a nanometer scale. For AFM, bacterial cells need to be firmly anchored to a substratum surface in order to withstand the friction forces from the silicon nitride tip. Different strategies for the immobilization of bacteria have been described in the literature. This paper compares AFM interaction forces obtained between Klebsiella terrigena and silicon nitride for three commonly used immobilization methods, i.e., mechanical trapping of bacteria in membrane filters, physical adsorption of negatively charged bacteria to a positively charged surface, and glutaraldehyde fixation of bacteria to the tip of the microscope. We have shown that different sample preparation techniques give rise to dissimilar interaction forces. Indeed, the physical adsorption of bacterial cells on modified substrata may promote structural rearrangements in bacterial cell surface structures, while glutaraldehyde treatment was shown to induce physicochemical and mechanical changes on bacterial cell surface properties. In general, mechanical trapping of single bacterial cells in filters appears to be the most reliable method for immobilization.