Environmental Engineering Reference
In-Depth Information
of random motility and chemotaxis from capillary assay data will be described
in Section 7.4.
Capillary assays are relatively easy to perform and have been widely used for
quantitative evaluation of chemotaxis. However, data obtained from this
method is not useful for relating population behavior to intrinsic cell para-
meters [18]. Mathematical models account for variability in setup geometry, but
since flow patterns at the mouth of the capillary are complex, such models are
based on simplified boundary conditions. This method is particularly suitable
for testing chemotaxis of sparingly soluble compounds, such as naphthalene [7].
7.3.2 Stopped-Flow Diffusion Chamber (SFDC) Assay
Figure 7.1B represents an experimental design for the SFDC introduced by Ford
et al. [18] for quantification of bacterial random motility and chemotaxis. This
assay is based on the principle that two liquid streams, with different chemoat-
tractant or chemorepellent concentrations, intersect via impinging flow to form a
step change in chemical concentration. Chemotactic bacteria respond either posi-
tively or negatively to the chemical gradient. This assay overcomes limitations of
the capillary assay by providing a well-characterized chemical concentration
gradient.
Briefly, bacteria that are uniformly distributed in the SFDC respond to a
chemical gradient induced by an initial step change in attractant concentration
and migrate toward optimal concentrations, resulting in a moving bacterial
band as time progresses (Fig. 7.1B). Light scattering microscopy is used to
Fig. 7.1 (continued) capillary tube that can be counted to quantify bacterial chemotaxis.
Bacterial accumulation at the mouth of capillary can also be observed under microscope.
SFDC assay (Panel B): Impinging flow is achieved by introducing two bacterial suspensions,
which differ in attractant concentration, into a channel formed between two microscopic
slides. After flow is stopped, temporal bacterial moving bands are imaged with light scattering
microscope and are converted into bacterial density vs spatial plots to quantify bacterial
chemotaxis. Agarose plug assay (Panel C): attractant is mixed with melted agarose plug and is
placed in the middle of a chamber formed by using cover slip and plastic strips at the top of a
microscopic slide. Bacterial suspension is flooded into chamber. Chemotactic band can be
visualized under microscope. Swarm plate assay (Panel D): Attractant mixed with growth
medium is poured into a Petri dish. Motile bacteria are stabbed in the middle. A sharp
chemotactic band moving outward from inoculation point can be observed (plate on left
side) in comparison to blank (plate on right side) which did not have any attractant with
growth medium. Tracking microscope assay (Panel E): Individual bacterium random walk is
tracked with tracking microscope to determine average run length and turn angle which in
turn are used to calculate the random motility and chemotactic sensitivity coefficients. [Note:
Pictures in Panels A, B, C (partially) D and E are adopted from Parales and Harwood (2002),
Ford et al. (1991) and Lewus and Ford (2001), Parales et al. (2000), Samanta et al. (2000) and
Ford and Harvey (2007), respectively.] Pictures in Panel B reprinted with permission of
Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.
