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Applied and Environmental Microbiology, July 2001, p. 3188-3194, Vol. 67, No. 7
División Corrosión,
INTEMA-CONICET, Universidad Nacional de Mar del Plata, B7608FDQ Mar
del Plata, Argentina
Received 8 January 2001/Accepted 16 April 2001
The adhesion of Pseudomonas fluorescens (ATCC 17552)
to nonpolarized and negatively polarized thin films of gold was studied in situ by contrast microscopy using a thin-film electrochemical flow
cell. The influence of the electrochemical potential was evaluated at
two different ionic strengths (0.01 and 0.1 M NaCl; pH 7) under
controlled flow. Adhesion to nonpolarized gold surfaces readily
increased with the time of exposition at both ionic-strength values.
At negative potentials ( Adhesion of bacteria to solid
surfaces is a general phenomenon associated with numerous medical,
industrial, and ecological problems (4, 7, 8, 11, 19, 22).
In particular, the adhesion to metal surfaces is related, for example,
to the contamination of prosthetic and medical devices (7)
or to the localized corrosion failure of industrial equipment
(4), as a consequence of the bacterial surface
colonization and biofilm formation. A better understanding of the
variables governing bacterial adhesion to metal surfaces will surely
contribute to funding solutions to these problems.
The interaction between bacterial cells and solid surfaces is often
described by the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory of colloid stability, developed by
Derjaguin and Landau in 1941 and Verwey and Overbeek in 1947 (14). This theory summarizes the electrostatic and van der
Waals interactions, yielding the overall interaction energy between
surfaces as a function of separation distance. In addition to these
nonspecific interactions, specific interactions have been described
participating in the bacterial adhesion process, including the
formation of ionic, hydrogen, and chemical bonds (3).
It was been pointed out that a reliable study of bacterial adhesion
requires well-defined hydrodynamic conditions and must prevent
alteration of results by avoiding the passage of samples through the
air-liquid interface (15, 16). These experimental constrains have been often circumvented while studying the adhesion of
bacteria to glass using both flow cell systems with well-defined flow
conditions and an in situ technique for the observation of the
interface (13). Following the same strategies, in
the present work we developed an experimental system which allows the
in situ observation of bacterial adhesion to metal surfaces by
phase-contrast microscopy and under controlled flow conditions.
Furthermore, since the system includes the possibility of an
electrochemical control over the metal surface, we studied the
influence of electrochemical variables on bacterial adhesion.
The objective of this work was to evaluate the influence of the
electrochemical potential on the bacterial adhesion to metals. Gold was
selected for the experiments as a model surface because it is a widely
studied noble metal and most of its physicochemical constants are
available in the literature (1, 20). In addition, the gold
surface composition remains constant over a wide potential interval,
and only changes of the electrostatic surface charge due to the
capacitive behavior of the electrical double layer take place
(18).
In this work, the existence of both reversible and irreversible
adhesion was demonstrated. Experimental results were compared with DLVO
calculations of the interaction energy at various potential and ionic
strength values.
Biological material.
Pure cultures of Pseudomonas
fluorescens (ATCC 17552) were grown at 32°C with continuous
shaking in a rich broth containing Lab Lemco (Merck) (0.1 g
liter Electrophoretic mobility.
The zeta potential ( Electrochemical thin-film flow cell.
A thin-film
electrochemical flow cell was especially designed for observation with
an optical microscope. A schematic representation of the cell is shown
in Fig. 1. It was constructed from a 75- by 35- by 3-mm acrylic piece and contained a shallow (2-mm) central chamber of 14 mm in diameter, through which the bacterial suspension was pumped. The working electrode (WE) was used as a lid for the chamber. It was constructed by sputtering a thin film (10 to 20 nm) of
gold onto a glass coverslip. The coverslip was previously degreased
with chloroform, cleaned by immersion in chromic acid, and washed
gently with double-distilled water and ethanol. The integrity of gold
films was verified by microscopic observation and their electric
resistance was measured in order to ensure electric conductivity.
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.7.3188-3194.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Adhesion of Pseudomonas fluorescens
(ATCC 17552) to Nonpolarized and Polarized Thin Films of Gold
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
0.2 and 0.5 V [Ag/AgCl-KCl
saturated {sat.}]), on the other hand, bacterial adhesion
was strongly inhibited. At 0.01 M NaCl, the inhibition was almost total
at both negative potentials, whereas at 0.1 M NaCl the inhibition was
proportional to the magnitude of the potential, being almost total at
0.5 V. The existence of reversible adhesion was investigated by
carrying out experiments under stagnant conditions. Reversible adhesion was observed only at potential values very close to the potential of
zero charge of the gold surface (0.0 V [Ag/AgCl-KCl sat.]) at a high
ionic strength (0.1 M NaCl). Theoretical calculations of the
Derjaguin-Landau-Verwey-Overbeek (DLVO) interaction energy for
the bacteria-gold interaction were in good agreement with experimental
results at low ionic strength (0.01 M). At high ionic strength (0.1 M),
deviations from DLVO behavior related to the participation of specific
interactions were observed, when surfaces were polarized to negative potentials.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
1), yeast extract (Sigma) (0.2 g
liter1), and peptone (Sigma) (0.5 g
l1) dissolved in 0.1 M NaCl, pH 7. Cells were
harvested from cultures at the exponential phase of growth by
centrifugation for 10 min at 10,000 × g in a Jouan
BR4i refrigerated centrifuge, washed with 0.1 M NaCl (pH 7), and
suspended in NaCl solutions of different ionic strengths (0.01 and 0.1 M NaCl, pH 7) after being centrifuged again.
) of
bacterial cells suspended in NaCl solutions (0.01 and 0.1 M) at a final
cell number of 105 cells
ml1, was determined by electrophoretic
migration. A Rank Bros., Ltd., Mark II particle microelectrophoresis
apparatus (Bottisham, Cambridge, England) was used. The applied
potential was 80 V. The value of was calculated using
Smoluchowski's equation as = 12.87 µ (5),
where µ is the measured electrophoretic migration.
View larger version (17K):
[in a new window]
FIG. 1.
Schematic diagram of the thin film electrochemical flow
cell designed for microscopic observations. Bacterial suspension is
pumped into the chamber through stainless steel liquid inlets (L in and
L out) at a controlled flow. The thin-film gold WE is placed facing
down and microscopic observations are made from the back. A platinum
wire CE circumvents the light path on the bottom of the chamber to
ensure a uniform current distribution. The reference electrode (RE) is
connected through a salt bridge to improve conductivity.
1, controlled by a low-flow peristaltic
pump. A pulse dampener was used to avoid pulsation in the liquid.
Adhesion measurements.
Once the cell was assembled and
filled with NaCl solution, the working electrode was polarized at the
desired potential using an EG&G 362 scanning potentiostat (Princeton
Applied Research, Princeton, N.J.). An Ag/AgCl-KCl saturated (sat.)
electrode was taken as the reference. Measurements were done at
potentials of 0.5 and 0.2 V, at the open circuit potential
(Eoc= 0.2 V) (Ag/AgCl-KCl sat.), and at two
different ionic strength values, 0.01 and 0.1 M NaCl.
1 was pumped into the chamber,
and cell adhesion to the working electrode was recorded on video tape
during 15 min. Sequential gray-scale images were captured from the
video tapes at 30-s time intervals to determine the kinetics of
bacterial adhesion under the different experimental conditions.
The digital analysis of images was done with the public domain NIH
Image software, developed at the National Institute of Health, which
can be obtained from http://rsb.nih.info.gov/nih.image/.
Every image was divided by the previous one and multiplied by 255 in
order to obtain, time to time, a resulting image containing only the
newly adhered bacteria. Out-of-focus moving bacterial cells were
clearly distinguished and ignored during the analysis process. Using
this process, newly attached cells appear as black (gray value, 255)
spots on the background color. On the other side, detachment can be
recognized by the appearance of white (gray value, 0) spots.
To determine the occurrence of reversible adhesion, additional
measurements were carried out under stagnant conditions, at the same
surface potentials previously used (0.5 V, 0.2 V and Eoc [Ag/AgCl-KCl sat.]). An additional surface
potential value of 0.00 V (Ag/AgCl-KCl sat.) was also included. In
these experiments, the bacterial suspension was manually injected with
a disposable syringe. Stagnant conditions were chosen in order to
improve the observation of bacterial movements in association with the
surface without interference from bacteria moving by the action of flow.
Sequential images were captured from the video records at a rate of 12 images s1 and analyzed with the NIH Image
software as follows.
Images (ni) were subtracted with the
previous one (ni1) to obtain
differential displacement images [d(t)] of
moving bacteria with 1/12-s time intervals. These subtractions yielded
differential images in which, beside the bacteria in their present
position at ni1, the position of
the bacteria at ni appears as white
footprints. Differential images [d(t = 1/12), d(t = 2/12) . . . d(t = n/12)] were compiled, and
the minimal gray value for every pixel during a 5-s (60/12-s) time
period was selected. This selection yielded a new image containing the (white) footprints of moving bacteria at every time interval. The
resulting image was multiplied by the last differential image [d(t = 60/12)] to include bacteria at their
present position in the result of the analysis.
Calculations of interaction forces between bacteria and gold
surfaces.
The interaction energies
[GDLVO(h)] between the
bacterial surfaces and the gold surfaces were calculated according to
the DLVO theory of colloidal stability with the mathematical equations in reference 14.
GDLVO(h) is the energy
resulting from the sum of van der Waals and electrostatic interactions.
The van der Waals attraction is proportional to the Hamaker constant
for the interaction between bacteria and gold across water
[Abg(w)] and was
calculated from the values of the Hamaker constants for each material
(14):
|
(1) |
) was used instead
of the electric potential, with the assumptions that the bacterial
surface is flat and that the charge is located on the plane of shear
(5, 21). However, it should be noted that the distance
h between a flat gold surface and the plane of shear of a
bacterial cell is a very approximate value.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Adhesion of P. fluorescens (ATCC 17552) to
nonpolarized and polarized gold at 0.01 M NaCl. To determine the
kinetics of bacterial adhesion to gold without any external
modification of the surface electrochemistry, experiments were
performed using bacterial suspensions in 0.01 M NaCl and with the gold
surface at its open circuit potential (Eoc=
0.2 ± 0.02 V [Ag/AgCl-KCl sat.]). Results can be observed in
Fig. 2. The number of adhered bacteria
readily increased with the time of exposition to the flowing bacterial
suspension, reaching 4.5 × 104 bacteria
mm2 after 15 min. Under these experimental
conditions, the spontaneous and irreversible adhesion of all the cells
reaching the surface was observed. When the surface potential was
externally controlled at a negative value, a strong inhibition of the
bacterial adhesion kinetics was observed. Both at 0.2 and 0.5 V
(Ag/AgCl-KCl sat.), the number of adhered bacteria was extremely low,
0.75 × 104 and 0.4 × 104
bacteria mm 2, respectively, compared to the
one obtained at the Eoc (Fig. 2).
|
, 0.2 V
(Eoc); 
, 
, Adhesion of P. fluorescens (ATCC 17552) to
nonpolarized and polarized gold at 0.1 M NaCl.
The effect of
increasing the ionic strength on the electrochemical potential
influence on bacterial adhesion was investigated. The experiments
described above were repeated, using in this case bacterial suspensions
in 0.1 M NaCl solution. The results are shown in Fig.
3. The higher number of adhered bacteria
was observed once again with the metallic surface at the
Eoc. After 15 min of exposition, about 2.5 × 104 bacteria mm2 were
adhered. This number was relatively lower than the one obtained at 0.01 M (4.5 × 104 bacteria
mm2) (Fig. 2).
|
0.2 V (Ag/AgCl-KCl sat.), about 1.5 × 104 bacteria mm2 were
adhered (Fig. 3), this number being relatively higher than the one
observed at 0.01 M NaCl (0.75 × 104
bacteria mm2) (Fig. 2).
The bacterial adhesion at 0.5 V (Ag/AgCl-KCl sat.) was negligible
(Fig. 3), as in the previous experiments under this condition (Fig. 2).
Calculations of the DLVO interaction energy curves.
Interaction energy curves were calculated for the DLVO interactions
between gold and bacteria in water. Values of 62.5 kT (1), 15.3 kT (17), and 9.34 kT (17) were used for the Hamaker constants of
the gold surface, the bacterial surfaces, and the surrounding water,
respectively. The surface potential for bacterial cells was
approximated to the value of their electrokinetic or zeta potential
() (21). The values of used in the calculations were 0.0315 and 0.0155 V at 0.01 and 0.1 M NaCl, respectively. These values were obtained from the electrophoretic migration (µ)
measured values, as equals 12.87 µ (5).
g) during the experiments, it was
necessary to take into account the PZC of the metal surface. Values
ranging from 0.00 to 0.07 V (Ag/AgCl-KCl sat.) were found in the
literature (20). The g values
used in the DLVO calculations were 0.57, 0.27, and 0.13 V, when
modeling the system at the surface experimental potentials of 0.5 V,
0.2 V, and Eoc (Ag/AgCl-KCl sat.), respectively.
The calculated interaction energy between gold and bacteria in 0.01 M
NaCl at various surface potentials as a function of separation distance
(H) can be seen in Fig. 4.
With the gold surface at its Eoc (0.13 V versus
the PZC), the interaction energy was strongly attractive, ranging from
43 kT at a separation distance of 20 nm to a primary
minimum deeper than 1,800 kT at distances of less
than 4 nm. On the basis of these calculations, irreversible adhesion
would be expected at this potential.
|
0.27 V and 0.57
V (versus the PZC), positive values of interaction energy in the liquid
reached up to 20 nm. Values higher than 100 kT at separation
distances of 10 to 15 nm determined the existence of extremely high
energy barriers against bacterial adhesion as a consequence of the
electrostatic repulsion.
At a higher ionic strength (0.1 M NaCl), the electrostatic interactions
were shielded at separation distances larger than ~5 nm (Fig.
5). Calculations with the gold surface at
Eoc resulted again in a primary minimum,
indicating the possibility of irreversible adhesion. Nevertheless, the
energy values in the proximity of the surface (<10 nm), were
significantly higher (less negative) than those calculated at low ionic
strength (Fig. 4), as a consequence of the diminution of the
electrostatic attraction.
|
82.9 and 94.9 kT at separation distances of 7 and 6 nm, respectively, when potentials of 0.57 and
0.27 V were used. The interaction was strongly repulsive at distances
shorter than 5 nm from the surface at both potentials (Fig. 5).
Reversible adhesion of P. fluorescens (ATCC 17552) to polarized gold. Calculations of the interaction energy between bacteria and gold at a high ionic strength (0.1 M NaCl) determined the existence of secondary energy minima when the surface was negatively polarized (Fig. 5). These secondary minima have been associated with the occurrence of reversible adhesion (3, 12, 14). In addition, the reversible adhesion of a Pseudomonas sp. to negatively polarized metal surfaces has been previously demonstrated (2). Since the experimental setup allows the direct observation of the behavior of bacteria in the polarized metal-electrolyte interface, experiments were carried out to verify if reversible adhesion of bacteria to gold occurs at the various potential and ionic strength conditions previously used. New sets of experiments were carried out in the absence of flow.
At low ionic strength (0.01 M), adhesion was detected only with the surface at its Eoc, as in the experiments using flow (Fig. 2). Once the bacterial cells approached the surface and adhered, no movements were observed. The irreversibility of the adhesion was proven at the end of the experiments by flowing distilled water through the chamber. The number of detached bacteria determined by digital image analysis was zero, indicating that cells adhered irreversibly to the gold surface in these conditions. At high ionic strength (0.1 M), different degrees of adhesion to the gold surface were detected at the various potentials (data not shown), resembling the results obtained using flow (Fig. 3). The adhesion to the surface at its Eoc was high and irreversible, as predicted by the DLVO interaction energy calculations (Fig. 5). On the other hand, the adhesion to the surface polarized to negative potentials (0.5 and 0.2 V [Ag/AgCl-KCl sat.) was low, as shown in
Fig. 3, but was also irreversible, in contrast to the presence of the
secondary minima predicted by the DLVO calculations (Fig. 5). Although
some bacterial cells described rotational movements during a few second
fractions when attaching to the surface, no conclusive evidence for
reversible adhesion was collected at these potentials.
To further investigate the occurrence of reversible adhesion,
additional experimental determinations were made with the gold surface
at a potential of 0.00 V (Ag/AgCl-KCl sat.), very close to the PZC
potential of gold (20). Reversible adhesion was clearly observed at this potential. Video records were digitally decomposed, and sequential images were analyzed. The results shown in Fig. 6 showed that some bacteria moved rapidly
in a plane parallel to the surface for a few seconds, describing a
random walk in a clear association with the surface. During the
translation, such bacteria occasionally sorbed at a pole, rotated in a
propeller-like movement (Fig. 6), and then broke away. Some other
bacteria just sorbed at a point and rotated before they broke away
(Fig. 6). These kinds of movements have been described by other authors in previous papers (6, 12) in relation to the occurrence of reversible adhesion to glass. Irreversible adhered bacteria were
also present during these experiments but were excluded from the
results as a consequence of the differential character of the digital
image analysis.
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133.1 kT located very close to the gold surface (4 nm) when a potential of 0.07 V
(versus the PZC) was used, as shown by DLVO calculations (Fig. 7).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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When a metal is immersed in an electrolyte solution, a potential
difference is immediately established between them, with the formation
of an electrical double layer. This double layer is composed by an
excess of charge on the metal surface (
M), separated from an excess of the opposite charge on the solution side
(S) by a layer of adsorbed water molecules.
The interfacial excess of charge on the solution side is a consequence of the accumulation of ions and is distributed towards the bulk solution forming a diffuse layer. The thickness of the diffuse layer
(i.e., the protrusion of the charge to the bulk solution) is dependent
on the solution ionic strength (18, 21). On the other
side, the interfacial excess of charge on the metal surface is
dependent on the surface electrochemical potential and can be modified
externally by polarization. It follows that if the potential of the
surface is shifted in the positive or negative direction, the charge
M increases in positive or negative value.
The potential at which the interfacial charge is zero
(M = S = 0) is
called the PZC (18).
Nonpolarized gold surfaces exhibit an open circuit potential of
0.2 ± 0.02 V (Ag/AgCl-KCl sat.) in NaCl solution, which is positive to the PZC of the material in the same electrolyte (0.00 0.07 V [Ag/AgCl-KCl sat.]) (20). The positive
interfacial charge of the metal surface at this potential determines
the electrostatic attraction against negatively charged bacteria,
which, in addition to the van der Waals attraction forces, results in a
deep primary minimum for the interaction energy, ranging from distances
larger than 20 nm (Fig. 4 and 5). As predicted by the DLVO calculations shown in Fig. 4 and 5, the observed adhesion at this potential was
irreversible under both ionic strength conditions and increased with
the time of exposition. The number of adhered bacteria was higher at
low ionic strength (Fig. 2) in accordance with the increased electrostatic attraction resulting from the decrease in shielding of
the positive charge at the gold surface (Fig. 4). Since virtually all
the cells which arrived at the surface adhered instantaneously, the
rate of the adhesion process in these experiments was assumed to be
dependent on the transport of cells to the surface.
Results shown in Fig. 2 and 3 indicated that bacterial adhesion to the
gold surface can be significantly affected by polarization to negative
potentials. The polarization to both, 0.5 and 0.2 V (Ag/AgCl-KCl
sat.), imposed a strong negative charge to the metal surface, which
resulted in a repulsive electrostatic interaction with the negatively
charged bacterial cells. As was shown by the DLVO calculations, at low
ionic strength (0.01 M NaCl) the electrostatic repulsion was increased
(Fig. 4), protruding beyond 20 and 15 nm into the bulk solution at
surface potential values of 0.57 and 0.27 V (versus the PZC),
respectively. This should be the reason why the number of adhered
bacteria to gold surfaces polarized to these potentials was strongly
reduced (Fig. 2).
The thickness of the interfacial double layer and, in consequence, the
influence of the electrostatic repulsion on the bacterial adhesion
process are dependent on the ionic strength. When ionic strength was
increased to 0.1 M NaCl, the interaction energy between negatively
polarized gold electrodes and bacteria was found to be repulsive at
shorter distances and became attractive at distances larger than ~5
nm (Fig. 5). The shielding of electrostatic repulsion can
explain the slightly increased bacterial adhesion at high ionic
strength observed at a potential of 0.2 V (Fig. 3), in relation to
the one observed at the low ionic concentration at the same potential
(Fig. 2). At a more-negative potential (0.5 V), however, the adhesion
remained very low (Fig. 3), probably due to the higher levels of
interaction energy at critical distances of 4 to 5 nm.
The overall interaction energy in 0.1 M NaCl (Fig. 5) presented
relatively deep secondary minima at 7 and 6 nm from the gold surface,
when potentials of 0.57 and 0.27 V, respectively (versus PZC) were
used. In spite of the presence of these minima very close to the
surface, the observed adhesion was irreversible, and no evidences of
reversible adhesion could be collected when experiments were carried
out at these negative potentials in the absence of flow.
The discrepancy between experimental and calculated results could be related to the participation of specific interactions (3). Also, the gold surface could be conditioned as a consequence of the adsorption of excreted metabolites, and surface properties could be slightly altered. However, the constancy of adhesion slopes shown in Fig. 2 and 3 indicates that conditioning of the initially clean surface does not occur during the experiments or does so at a minor rate.
Regarding specific interactions, it was proposed that bacteria adhere to the surface of minerals (10) and metal oxides (J. P. Busalmen and S. R. de Sánchez, submitted for publication) through the formation of hydrogen bonds. Gold surfaces support covalent bonding with water molecules in the molecular state in order to compensate for the excess of surface charge. Since the energy of these covalent bonds is close to the energy of hydrogen bonds, adsorbed water molecules are combined not only with the metal surface but also with water and other molecules by hydrogen bonding (18). The excess of surface charge on a metal surface gradually disappears with the formation of succeeding layers of water molecules. On the other side, it was emphasized that DLVO interactions of bacterial cells more likely originate from a deeper shell in the cell core, other than at the cell surface, and that the projection of a few lipopolysaccharide molecules could anchor the cell irreversibly to the surface (9).
The width of a water layer is about 0.25 nm, and the formation of several layers at surface potentials where the surface charge density is high (far from the PZC) could bridge the distance to the bacterial cells at the secondary minimum, allowing the formation of hydrogen bonds with the lipopolysaccharide on the bacterial surface and yielding irreversible adhesion in spite of energy barriers.
The absence of reversibly adhered bacteria could be related to both phenomena: the quick change to irreversible adhesion through the formation of hydrogen bonds as soon as bacteria reached the secondary minimum and the electrophoretic migration of negatively charged bacteria far from the gold surface along the electric field generated in a direction perpendicular to the surface, as a consequence of the potential difference between the WE and the CE.
Reversible adhesion was observed only when the gold surface was polarized to a potential very close to its PZC (0.00 V [Ag/AgCl-KCl sat.]). Due to the low overpotential, the surface charge was very low and the electrostatic repulsion was reduced to distances shorter than ~2.5 nm (Fig. 7). In contrast to the situation on a strongly polarized surface, neither the organization of water layers nor the presence of an electric field is significant enough to interfere with the presence of bacteria reversibly sorbed at the secondary minimum. In addition, the depth of the secondary minimum was enough to retain bacteria very close to the surface (4 nm) but allow movements in the parallel plane as shown in Fig. 6.
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ACKNOWLEDGMENTS |
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The present research was supported by a grant (PIP 4339) from CONICET-Argentina.
Useful discussions with A. Regazzoni are gratefully acknowledged.
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FOOTNOTES |
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* Corresponding author. Mailing address: División Corrosión-INTEMA, UNMdP, Juan B. Justo 4302, B7608FDQ Mar del Plata, Argentina. Phone: 54 223 4816600. Fax: 54 223 4810046. E-mail: jbusalme{at}fi.mdp.edu.ar.
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Applied and Environmental Microbiology, July 2001, p. 3188-3194, Vol. 67, No. 7
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.7.3188-3194.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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