Biology Reference
In-Depth Information
physiological factors such as growth rate and culture conditions. Micrographs can be
automatically analyzed using imaging software such as ImageJ (
Abramoff
et al.
,
2004
) and the distribution of cell length and diameter determined automatically
by using the Feret's diameter property of all selected image features (i.e. bacterial
cells). The most precise measurement of cell volume is likely to be accomplished
using membrane stains that facilitate the calculation of the true intracellular volume.
3.1.5
Cell disruption
Cell disruption is crucial for the release of intracellular components such as nucleic
acids, metabolites or proteins. The cell disruption method that is used must be effec-
tive in releasing these cellular compounds reliably, efficiently and effectively. In the
context of systems biology, special attention has to be paid to disrupting the cells in a
very short-time frame, otherwise, the analyses will be biased by effects associated
with the analysis workflow rather than the physiology of the cells during cultivation.
Additionally, because of the labile nature and the dynamics of biological macromol-
ecules, the sample temperature should be kept to a minimum, ensuring cooling to
4
C or below during the entire cell disruption process.
A detailed analysis of bacterial cell disruption techniques reveals a clear corre-
lation between disruption efficiency, the species being investigated (including cell
wall type) and the physiological state of the cells. Rod-shaped bacteria are easier
to disrupt than coccal-shaped cells that form a vault-like structure that is much more
resistant to mechanical forces. The cell walls of Gram-positive bacteria are
more rigid than those of Gram-negative bacteria, whilst wall-less bacteria (e.g.
Mycoplasma
) can usually be disrupted by hypo-osmotic shock. Stabilizing or
surrounding layers such as surface (S) layer proteins and polysaccharide capsules
may influence cell disruption efficiency. Moreover, it is known that the Gram-
positive bacteria
B. subtilis
and
S. aureus
thicken their cell walls upon entry into
stationary phase, resulting in reduced disruption efficiencies compared to exponen-
tially growing cells (
Middelberg, 1995
). Also, younger (i.e. newly born) cells may be
disrupted more easily than older ones. Finally, the disruption process is tightly con-
nected to the system under investigation and needs to be optimized for any species,
cell type and cell state. As shown in
Figure 3.2
, we have tested a variety of cell lysis
methods for both
B. subtilis
and
S. aureus
in order to identify the most effective
workflow for both exponentially growing and stationary phase cells. By using the
optimized parameters, disruption efficiencies
90% are achievable. However, it
is becoming increasingly clear that cell disruption technology still needs to undergo
substantial improvements to ensure efficiency and lack of discrimination for cells of
differing sizes, shapes and composition.
To ensure efficient and complete cell lysis of each specific cell type, it is advisable
to evaluate all themethods available in the respective laboratories. Benchmarking cell
disruption efficiencies may be based either on the determination of colony-forming
units or on the counting of intact cells in a counting chamber. Furthermore, cell
viability assay based on, for example, fluorescence markers may also be used.
