Hj Busscher

The Influence of Surface Free-energy on Planimetric Plaque Growth in Man

The purpose of this study was to examine the change in plaque area over nine days in vivo on four materials with different surface free-energies (s.f.e.). Twelve healthy dental students participated in a crossover, split-mouth, double-blind study. Supragingival plaque formation was recorded over a nine-day period, on four different materials: fluorethylenepropylene (Teflon) (FEP), parafilm (PAR), cellulose acetate (CA), and enamel (E) with s.fe. of 20, 26, 57, and 88 erg/cm2, respectively. Strips made from the first three materials were stuck to the buccal surface of an upper incisor. The remaining incisor was carefully polished and served as an enamel surface. The increase in plaque was evaluated after three, six, and nine days. A planimetrical analysis was used so that the plaque area could be expressed as a percentage of the total buccal tooth surface. This procedure was repeated on each subject, so that at the end, each pair of central or lateral incisors received the four tested materials. The results indicated that the adherence of micro-organisms on pellicle-coated substrata was influenced by the materials s.f.e.; there was an association between the s.f.e. of the substrata and the supragingival plaque extension in vivo. High surface free-energy substrata in the oral cavity attracted more micro-organisms than did low energetic materials. Additionally, the bacterial adhesion seemed very weak on surfaces with a low s.f.e.

Properties of oral streptococci relevant for adherence: Zeta potential, surface free energy and elemental composition

Abstract In order to characterize the functional properties of the cell surface relevant for adherence, the zeta potentials, surface free energies and elemental surface compositions of oral streptococci were determined. Zeta potentials of fully hydrated cells were measured as a function of pH in dilute potassium phosphate solutions, yielding isoelectric points (IEP) for the different strains. Surface free energies were calculated according to the geometric mean equation from contact angles, measured on bacterial cells deposited on a membrane filter. The elemental analyses were carried out on freeze-dried cells by X-ray photoelectron spectroscopy. A decrease in the N/C concentration ratio among the different strains from 0.124 (S. mitisBMS) to 0.053 (S. salivarius HBC12) was found to be concurrent with an increase in the O/C concentration ratio from 0.312 to 0.495. Simultaneously, the IEP shifted from 3.7 to 1.3 and the surface free energy changed from 37 to 125 mJ m−2. The zeta potentials, the surface free energies and the elemental compositions of the bacterial cell surface showed clear relationships despite the fact that these parameters were measured in different states of hydration of the surface.

MICROBIAL FACTORS IN A THERMODYNAMIC APPROACH OF ORAL STREPTOCOCCAL ADHESION TO SOLID SUBSTRATA

Abstract In this paper surface modification of bacterial adhesion sites on glass by excreted macromolecular substances from Streptococcus mitis BMS cells was investigated by probing the affinity of Streptococcus mutans NS cells for the glass surface. Excreted substances which were different from the fibrillar surface appendages of this strain affected the adhesion sites and hence the adhesive behavior of S. mutans and the producer strain itself. Such surface modifications caused by excreted substances can occur frequently but until now their effects have been neglected in a thermodynamic approach on bacterial adhesion to solid substrata. Recently we proposed that the number of bacteria adhering at equilibrium to solid substrate, n e , is linearly related to the interfacial free energy of adhesion according to n e = a (Δ F adh − 23.1) + 1.2 (I. H. Pratt-Terpstra, A. H. Weerkamp, and H. J. Busscher, Curr. Microbiol. 16 , 311 (1988)). In this relation a denotes a strain-specific microbial factor with a characteristic value for each strain which was associated with the absence or presence of fibrillar surface appendages. In the present study we showed that the low a factor of S. mitis BMS is due to surface modifications caused by its excretion of antiadhesive substances. Recalculations of data provided by various other authors confirmed these results, independent of whether the thermodynamic approach employed involved the equation of state or the dispersion and polar components concept. Therefore the inclusion of an a factor can be done in a general approach to bacterial adhesion to solid substrata. Tentatively this strain-specific a factor must be considered as a parameter because of its possible dependence on temperature, pH, ionic strength, and shear rate, but it is expected that this factor can be evolved to a purely strain-dependent constant by changing the variables and extending the above equation.

A QUANTITATIVE METHOD TO STUDY CO-ADHESION OF MICROORGANISMS IN A PARALLEL-PLATE FLOW CHAMBER - BASIC PRINCIPLES OF THE ANALYSIS

Intermicrobial aggregation is described as one of the factors contributing to dental plaque formation. Intermicrobial aggregation is usually measured by mixing potential partners suspended in a liquid phase (‘coaggregation’). However, even if aggregation in the liquid phase occurs, adhesion of microorganisms to partners already adhering to a substratum surface may also occur (‘co-adhesion’). Coaggregation assays have been performed in order to measure coaggregation and to model co-adhesion, although it is not yet clear which the two prevails in vivo. Apart from being semi-quantitative (scores run from 0 to 4) it is questionable whether coaggregation assays really mimic co-adhesion. This study was designed to develop a method to quantitative assess co-adhesion of microbial pairs in order to gain a better understanding of the mechanisms governing co-adhesion. Co-adhesion of coaggregating and non-coaggregating partners (S. oralis, S. sanguis and A. naeslundii) to glass has been studied in a parallel plate flow chamber using real time image analysis. The spatial arrangements of adhering bacteria were analyzed by radial pair distribution functions, revealing the relative density of adhering bacteria around adhering bacteria from the same (g22(r)) or a partner strain (g21(r)). Pair distribution functions g21(r) of coaggregating pairs clearly reveal a preference of coaggregating streptococci (S. oralis J22 and S. sanguis PK2951) to adhere around the actimomyces (A. naeslundii PK213 or T14V-J1), which were used to coat the bare glass substratum. Besides, the distribution function g21(r) showed differences in co-adhesion patterns for strains with the same coaggregation score. From the results presented in this paper it can be concluded that with a parallel plate flow chamber, co-adhesion can be quantified on a continuous scale under well controlled conditions, more closely resembling those occurring in vivo.

ASSESSMENT OF BACTERIAL BIOSURFACTANT PRODUCTION THROUGH AXISYMMETRICAL DROP SHAPE-ANALYSIS BY PROFILE

SummaryAxisymmetric drop shape analysis by profile (ADSA-P) is a technique developed in colloid and surface science to simultaneously determine the contact angle and liquid surface tension from the profile of a droplet resting on a solid surface. In this paper is described how ADSA-P can be employed to assess bacterial biosurfactant production. Nine Streptococcus mitis strains, two of which are known to produce biosurfactants, and two S. salivarius strains, which do not produce biosurfactants, were suspended at two concentrations in a 10-mm potassium phosphate buffer, pH 7.0. Subsequently, a 100-μl droplet of each suspension was put on a fluoroethylenepropylene surface and the profile of the droplet determined with a contour monitor as a function of time up to 2 h. The surface tension of these suspensions was then calculated from the droplet profiles with ADSA-P. The surface tension of suspensions of the two non-producing strains remained stable within 4 mJ·m−2, whereas the surface tension of suspensions of five out of the nine S. mitis strains employed, including those of the known producer strains, decreased significantly (up to 26 mJ·m−2). This decrease was, in addition, concentration dependent. From these observations, we decided that all strains for which these concentration-dependent decreases were observed, could be regarded as biosurfactant producers. In order to rule out the possibility that the surface tension decreases observed were due to the collection of cells at the suspension-air interface, we investigated whether there was a relationship between surface tension decrease and hydrophobicity of the cells, as assessed by contact angle measurements and bacterial adhesion to hydrocarbons. Since no such a relationship was found, it can be concluded that ADSA-P is an excellent technique, based on using small amounts of cells to rapidly determine whether or not a bacterial strain produces biosurfactants.

An Interlaboratory Comparison of Physicochemical Methods for Studying the Surface-properties of Microorganisms - Application To Streptococcus-thermophilus and Leuconostoc-mesenteroides

The overall properties of the cell surface of Streptococcus thermophilus and Leuconostoc mesenteroides, two fouling microorganisms from dairy industry, were determined by a variety of physico-chemical methods. The study was carried out as an interlaboratory comparison to identify seemingly unimportant but yet critical steps in the experimental methods, i.e., contact-angle measurements, electrophoresis, X-ray photoelectron spectroscopy (XPS) and transmission Fourier-transform IR spectroscopy (FTIR). Particularly with regard to the contact-angle measurements, it turned out that the original papers, in which the technique was explained, did not contain enough detail. On the basis of the present experience, more emphasis is put on a slow preparation of bacterial samples and monitoring of the drying process by contact-angle measurements. Furthermore, the known importance of the suspending buffer in zeta potentials becomes extremely obvious since one of the strains employed preferentially adsorbed K+ from the buffer. Artefacts created during freeze-drying for XPS and FTIR analyses are mentioned. It is pointed out that the above four methods form a unique set of techniques for studying microbial cell surfaces, in the sense that the results of the various methods allow an interpretation of the physico-chemical properties of the cells in terms of their chemical composition.

AGING EFFECTS OF REPEATEDLY GLOW-DISCHARGED POLYETHYLENE - INFLUENCE ON CONTACT-ANGLE, INFRARED-ABSORPTION, ELEMENTAL SURFACE-COMPOSITION, AND SURFACE-TOPOGRAPHY

Aging effects of repeatedly oxygen glow-discharged polyethylene surfaces were determined by water contact angle measurements, infrared (IR) spectroscopy, X-ray photoelectron (XPS) spectroscopy, and surface topography determination. Glow-discharged surfaces were stored at room temperature and in liquid nitrogen for 8 days prior to the next glow-discharge (Gld) treatment. This cycle was repeated up to 13 times. Hydrophobic recovery of the polyethylene surface, as determined by contact angle measurements, became less when the number of glow-discharge treatments increased. Hydrophobic recovery was suppressed when the samples were stored in liquid nitrogen. After five glow-discharge treatments it was possible to detect the incorporation of hydroxy groups by IR spectroscopy, while XPS spectroscopy showed that repeated glow-discharge gives rise to a higher oxygen to carbon ratio and a broadening of the C1s peak, suggesting the incorporation of C - O and C = O bonds in the surface layer of polyethylene. The surface roughness of repeatedly glow-discharge-treated polyethylene remained almost unaltered in the submicrometer range (0.30-0.35-mu-m), although scanning electron microscopy revealed fine-grained structures for samples after a large number of Gld treatments. It can be concluded from this study that repeated oxygen glow-discharge treatments of polyethylene create more stable, hydrophilic surfaces than can be obtained with only one treatment. However, even after repeated glow-discharge treatments, polymer surface dynamics cause a small hydrophobic recovery.

COMPARISON OF THE PHYSICOCHEMICAL SURFACE-PROPERTIES OF STREPTOCOCCUS-RATTUS WITH THOSE OF OTHER MUTANS STREPTOCOCCAL SPECIES

Mutans streptococci comprise a group of seven closely related, yet distinct species. The distinction between the four species used in this study, namely Streptococcus sobrinus, Streptococcus cricetus, Streptococcus rattus, and Streptococcus mutans, has been made only recently on the basis of DNA homologies. In order to determine if there is a difference in the physicochemical surface properties of these species, strains were characterized by contact angles, zeta potentials and isoelectric points (IEP), elemental surface compositions by X-ray photoelectron spectroscopy, and molecular moieties by infrared spectroscopy. Contact angles, particularly when measured with water, can be considered a measure of cell surface hydrophobicity; zeta potentials reflect the charge of the outermost cell surface; X-ray photoelectron spectroscopy yields the relative abundance of carbon, oxygen, nitrogen, and phosphorus over the outer 5 nm of the bacterial cell surface; infrared spectroscopy enables a molecular characterization in terms of proteins, phosphates, and polysaccharides. All four species were homogeneous with regard to their physicochemical surface properties. However, the S. rattus species were clearly different from the others on the basis of the low water contact angle (21 +/- 2 vs. 26-31 degrees), highly negative zeta potential and lack of IEP, and high oxygen/carbon (0.50 +/- 0.02 vs. 0.41-0.43) and phosphorus/carbon (0.016 +/- 0.001 vs. 0.006-0.008) surface concentration ratios. Amongst the other differences observed, each species had a characteristic pH dependence of their zeta potential measured in phosphate buffer, yielding an IEP of 1.7, 2.1, and 2.5 for S. cricetus, S. sobrinus, and S. mutans, respectively. However, a cluster analysis on the zeta potential data showed only an isolated cluster for the S. rattus species. Thus it is likely that the higher cariogenicity of S. sobrinus with respect to S. cricetus and S. mutans is, in addition to a higher acidogenicity, due to the smaller negative surface charge, giving an increased adhesion to negatively charged tooth surfaces. S. rattus is expected to be the least adherent strain in the oral cavity as it carries a relatively high negative surface charge.

Preparation and characterization of superhydrophobic FEP-Teflon surfaces

Superhydrophobic FEP-Teflon was prepared by argon ion etching followed by oxygen glow discharge treatment of commercially available FEP-Teflon sheet material. This combined treatment yielded an increase in water contact angle from 109-degrees to > 140-degrees. Ion etching alone caused a small increase in surface roughness and a loss of fluorine from the surface, but the water contact angles increased only to 120-degrees. Scanning electron micrographs of ion-etched surfaces showed stalky protrusions with a diameter of approximately 40 nm. Glow discharge treatment of ion-etched surfaces reduced the length of these protrusions and therewith the microscopic surface roughness. However, in all cases the macroscopic surface roughness was less than 1-mu-m (R(A) value). X-Ray photoelectron spectroscopy indicated major changes in elemental surface composition as a result of the treatments. These modifications did not influence the infrared absorption spectra (attenuated total reflection) of the modified surfaces, indicating that the chemical changes brought about are really superficial. It is concluded that the superhydrophobicity created is mainly due to changes in the specific, microscopic surface topography resulting from ion etching, but also partly due to the role of the glow discharge treatment, restoring a high surface concentration of fluorine after ion etching.

SURFACE FREE-ENERGY CHANGES OF HUMAN-ENAMEL DURING PELLICLE FORMATION - AN INVIVO STUDY

The surface free energy and its polar- and dispersion components pertaining to pellicle-covered human enamel, sintered hydroxyapatite, Diacryl® and fluorethy-lenepropylene were determined from contact angle measurements. During the first 5 min of pellicle formation, the total surface free energy of human enamel increased from 84 to 110 erg·cm––2. After these first 5 min the surface free energy of pellicle covered solids remained constant for at least 2 h. Pellicle formation on apolar solids like fluorethylenepropylene increased the surface free energy only slightly.