Biology Reference
In-Depth Information
on the washing solution. We acknowledge that successful application of this fast
filtration method requires some training, but in our experience, it remains the method
of choice for accurate metabolome sampling from bacteria in liquid culture of
low to moderate biomass concentration. For cultivation with high cell density
(
5g
DryWeight
L
1
), we recommend extracting the whole culture broth as described
in method B below.
The great advantage of fast filtration is the low contamination of the samples with
salts, which dramatically improves the separation of sugar phosphates and also the
sensitivity of the LC-MS. As a result, the number of quantifiable metabolites is much
higher when compared to whole cell broth extraction technique. A further advantage
is the complete removal of extracellular metabolites since the cells are continuously
perfused with a washing medium. The method only requires minimal technical
set-up as sampling is performed manually with a pipette.
Precaution
. Shake flask culture cells should be recovered from the same phase of
pseudo steady-state growth and the biomass concentration should be in a range of
OD
600
0.5-1 to prevent artefacts caused by oxygen limitation and the accumulation
of by-products. In our experience, a fast-growing bacterial culture (
>
0.6 h
1
)of
this biomass concentration depletes the oxygen dissolved in minimal medium within
1 min. The quantities of cells and volumes are given to detect metabolites with a
concentration of 1-100
m
mol L
1
in the LC-MS samples.
m
¼
3.1
Fast filtration
Fast filtration was initially proposed by
Wittmann
et al.
(2004)
, and we apply this
method to obtain steady-state metabolite concentrations in bacteria under robust
and stable growth conditions as described in
Section 2
(
Figure 5.1
):
Assemble a vacuum filter flask and preheat to the temperature used for
cultivating cells in a thermostatically controlled room.
Shake flask
culture
Fast filtration
Centrifuge
and store
supernatant
Wash with
growth medium
Load 2 mL
culture broth
on filter
OD
600
0.5-1
Quenching
and
extraction
FIGURE 5.1
Schematic of fast filtration. Culture broth from a shake flask is transferred onto a nitrocellulose
filter mounted on a vacuum device. Subsequently, cells are immediately washed with
growth medium. Following filtration and washing, the intracellular metabolites are extracted
directly from the cell-loaded filter disc and prepared for analysis.
