Towards understanding inter-strain attachment variations of Escherichia coli during transport in saturated quartz sand - Transport of Multiple Escherichia Coli Transport in Saturated Porous Media - page 32

test tube and inoculated at the centre of petri-dishes containing 0.35% Chromocult agar. The
plates were incubated at 37 °C for 24 hours after which growth and diameter of migration was
measured as motility (Ulett et al., 2006).
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 as reported by 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 determine auto-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 1cm 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 %).
3.2.5 Serotypes and lipopolysaccharide structure
Most of the information on polysaccharide structure of the various serotypes can be found in a
database on the Internet ( www.casper.organ.su.se/ecodab ) . The database is described in detail by
Stenutz et al. (2006). Since 2006, the polysaccharide structure of a limited number of additional
E. coli serotypes was elucidated, and included in this research.
3.2.6 Detection of genes encoding factors related to E. coli surface structures
The polymerase chain reaction (PCR) is a powerful technique to amplify a single or few copies
of a piece of DNA (here, genes possible involved in attachment) for several orders of magnitude,
generating millions or more copies of a particular DNA sequence. The method relies on thermal
cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting
and enzymatic replication of the DNA. Prior to PCR, bacterial cultures were pre-grown in
nutrient broth (OXOID CM 001) for 24 h at 37°C. DNA of E. coli cells was isolated with a
FastDNA Spin Kit (QBiogene), involving a mechanical 'bead-beating' procedure.
Qualitative PCR was carried out for the ten strains with highest attachment efficiencies and for
the ten strains with lowest attachment efficiencies in 48-well plates in a 25 L volume containing
1 µ l of isolated DNA, 1 µ l of both forward primer (10 µM) and reverse primer (10 µM), 12 µ l
FideliTaq PCR Master Mix 2× (USB Cooperation, Ohio, USA), and 10 µ l MilliQ water. PCR
conditions consisted of an initial denaturation step (4 min 94 °C), 25 cycles of denaturation (at
92 °C for 1 min), annealing (dependent on primer) and extension (72 °C for 1 minute), and a
final elongation cycle (5 min at 72 °C). Primers used are given in Table 3.1 . Primers prepared in
this study ( Table 3.1 ) were determined with PRIMERBLAST by using the E. coli genes as query
sequences ( www.ncbi.nlm.nih.gov ). PCR mixtures were electrophoresed on a 1.2% agarose gel
stained with ethidium bromide (0.08 g/ml) for 30 minutes at 80-100 V. Results were made
visible under UV light (302 nm) with a UV transilluminator.