Biology Reference
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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 254 nm.
To determine cell aggregation , 15 ml of freshly grown bacteria were centrifuged (14000 xg)
and washed three times in AGW, and, then, allowed to stand for 180 minutes at a temperature
of 4 °C. A sample of 1 mL 1 cm below the surface of the suspension was obtained,
immediately and 180 minutes after washing. The optical density of the samples was measured
at 254 nm, and the auto-aggregation was determined as the ratio of the final over the initial
optical density (in %).
To determine the zeta potential , 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., 2007).
Bacteria mobility values were obtained from measurements on at least 50 bacteria cells.
Velocity measurements were used to calculate the zeta potential with the Smoluchowksi
equation.
To measure the average equivalent spherical diameter 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. At least 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 was determined as the geometric mean of
average length and width (Rijnaarts et al., 1993).
7.2.6 Serotyping
Pure cultures of E. coli were grown on Chromocult agar and sent to the Dutch National
Institute of Public Health and Environmental Hygiene (RIVM), Utrecht, The Netherlands,
where serotyping was performed according to standard procedures. For O typing, the strains
were tested against O1 antiserum until O181 antiserum using the classical approach (Guinée
et al., 1972). For H typing, the strains were incubated 30°C and test performed in small glass
tubes against H antisera H1 until H56. Details of the method are described in Ewing (1986).
7.2.7 Porous media and transport experiments
The porous media comprised of 99.1% pure quartz sand (Kristall-quartz sand, Dorsilit,
Germany) with sizes ranging from 180 to 500 m, while the median of the grain size weight
distribution was 356 m. With this grain size, we excluded straining as a possible retention
mechanism in our column: assuming a bacteria equivalent spherical diameter of 1.5 m, the
ratio of colloid and grain diameter was 0.004, which was well below the ratio (0.007) for
which straining was observed by Bradford et al. (2007) for carboxyl latex microspheres with
a diameter of 1.1 mm suspended in solutions with ionic strengths up to 31mM (the ionic
strength of the solutions we used was 4.7 mmol/L only). Total porosity was determined
gravimetrically to be 0.39. The quartz sand was rinsed sequentially with acetone, hexane and
concentrated HCl, followed by repeated rinsing with de-mineralized water until the electrical
conductivity was close to zero (Li et al., 2004).
To assess the transport characteristics and attachment variations among E. coli strains
isolated after they had undergone transport in an aquifer, forty of the seventy-one E. coli
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