Environmental Engineering Reference
In-Depth Information
7.3.4 Swarm Plate Assay
This qualitativemethod is based onmetabolismof an attractant. A carbon source
(attractant) is mixed with low percentage agar media (generally 0.3% [7]) and
poured in a petri dish. Bacteria are stabbed in the middle of the petri dish and
incubated. A sharp chemotactic band of bacteria growing outward is visualized in
the petri dish as a result of attractant metabolization and the resultant concen-
tration gradient (Fig. 7.1D). This method is well suited for identification of
chemotactic bacteria and is widely used for enrichment of chemotactic mutants
[7, 22, 23, 24, 25]. However, as metabolism is a primary requirement, this method
is limited to testing chemotaxis of metabolizable chemoattractants [7].
7.3.5 Drop Assay
Similar to the swarm plate assay, this method is also based on population-scale
imaging and may be used to study bacterial responses to both chemoattractants
and chemorepellents. Chemotactic bacteria are suspended in a drop assay agar
consisting of bacto-agar (0.3%) and a carbon source (1 mM glucose, for
example) [25], and poured into a petri dish. The chemotactic response is
determined by placing an attractant/repellent in the center of the petri dish,
incubating, and observing the characteristic chemotactic band formed sur-
rounding the drop.
7.3.6 Three-Dimensional Tracking Microscopy
In addition to the population-based assays described above, individual cell
tracking assays for evaluating random motility and chemotaxis are also
described in the literature [15, 18, 26], and are based on a three-dimensional
tracking microscope developed by Berg [27]. This method consists of character-
izing the 3D random walk, described earlier in Section 2.2, of individual
bacteria (Fig. 7.1E). The parameters of interest are average run length (l),
average run time (
), and average turn angle (
) of the bacteria. The following
mathematical relationship, derived by Lovely and Dahlquist [26] and based on
statistical analysis of individual cell-based parameters, can be used to calculate
a population-based random motility coefficient.
0
ΒΌ
lv
3
1
1
cos
(7
:
2)
where v is the average chemotactic velocity and can be calculated from the
average run length and average run time.
