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was measured as cell hydrophobicity. Thereto, 4 mL of bacteria suspension of known optical
density and 1mL of dodecane were vigorously mixed in a test tube for two minutes and left to
stand for 15 minutes to allow phase separation. Then, the optical density of the aqueous phase
was determined, and the percentage of cells partitioned into the hydrophobic substance was
reported as percentage hydrophobicity. All optical densities were measured at an absorbance of
546 nm (Walker et al., 2005).
To determine the
outer surface potential
(OSP), a zeta-meter similar to the one made by Neihof
(1969) was used. Movement of bacteria was visible on a video screen attached to a camera
mounted on top of a light microscope (Olympus EHT) in phase contrast mode (Foppen et al.,
2007b). Bacteria mobility values were obtained from measurements on at least 50 bacteria cells.
Velocity measurements were used to calculate the OSP of the
E. coli
cells according to
Ohshima's electrokinetic theory for soft particles (Ohshima, 1994; Dague et al., 2006). To
determine the outer surface potential, we used the low potential approximation, given by De
Kerchove and Elimelech (2005):
1
1
+
Ρ
Η
Κ
Λ
fix
Ψ
=
Μ
−
(2.1)
0
2
1
2
ΗΛ
ΕΕ
+
r
0
Κ
Λ
whereby
Μ
is the eletrophoretic mobility (m
2
V
-1
s
-1
),
Ψ
is the OSP of an
E. coli
cell (V),
fi
Ρ is
the fixed charge density of the polyelectrolyte layer at the
E. coli
surface (mol),
Η
is the fluid
dynamic viscosity (Pa s),
1
/
Λ
is the electrophoretic softness (m),
Ε
is the dimensionless
dielectric constant,
Ε
is the dielectric permittivity in a vacuum (CV
-1
m
-1
), and1/
k
is the double
layer thickness (m). For the calculations, we assumed the electrophoretic softness to be 0.74 nm
-1
and the fixed charge density to be -150 mM. These values were taken from De Kerchove and
Elimelech (2005) for an
E. coli
strain (D21g) with a soft layer of lipopolysaccharides in the outer
membrane.
To determine
motility
, a cell suspension in cow manure extract was filtered through 0.2 µ m
cellulose acetate membrane filter, and by means of a sterile toothpick, cells were picked from the
filter membrane and inoculated at the centre of petri-dishes containing 0.35% agar (Oxoid agar
technical-agar no. 3), supplemented with manure extract. The plates were incubated at 23 °C for
48 hours after which growth and diameter of migration was measured as motility (Yang et al.,
2006; Ulett et al., 2006).
To determine
width
and
length
of the cells, a light microscope (Olympus BX51) in phase contrast
mode, with a camera (Olympus DP2) mounted on top and connected to a computer, was used to
take images of cells. Averages of 50 images were imported into an image processing program
(DP-Soft 2) and the average cell width and cell length were measured. The equivalent spherical
diameter (ESD) was determined as the geometric mean of average length and width (Rijnaarts et
al., 1993), while the cell sphericity was obtained from the ratio of average width to average
length (Weiss et al., 1995).
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