Biology Reference
In-Depth Information
Prior to sampling determine the OD and pH of the culture.
Prepare 100 mL cultivation media to wash the cells, adjust to the pH of the culture
at the time of sampling and preheat to the temperature used for cultivating the cells.
Place a 0.45-
m pore size filter with 25 mm diameter (e.g. Millipore, no.
HAWP02500) on the vacuum filter flask and apply a vacuum of
m
50 mbar.
Pre-wash the filter with medium such that it is completely wetted.
Transfer 2 mLof the culture broth onto themiddle of the filter using a pipette. Load
the filter carefully but be fast enough to prevent oxygen limitation in the pipette.
Perfuse the filter with 5 mL of the preheated washing mediumwhile ensuring that
the filter never runs dry during loading and washing. The medium absorbed in the
filter will provide nutrients only for a few seconds. Assure that the washing
medium resembles conditions in the culture broth. Any modification of the
washing medium can cause artefacts.
After washing, immediately transfer the cell-loaded filter to the extraction
solution using a pair of tweezers. The extraction solution will arrest metabolic
activity and release metabolites from the cells (see
Section 3
).
3.2
Whole cell broth extraction
Fast filtration is not applicable if environmental conditions are likely to change
during filtration, for example, when sampling from bioreactors with high-biomass
concentrations and controlled substrate concentrations. In this case, we recommend
extracting the whole culture broth and applying a differential method to quantify
intracellular metabolites (
Taymaz-Nikerel
et al.
, 2009
). A major drawback is the
contamination of the sample with salt from the medium, which causes reduced
sensitivity and thus may reduce the number of metabolites that can be quantified.
Furthermore, it is not possible to discriminate between the intracellular and extracel-
lular location of a metabolite prior to extraction. However, in case of high biomass
concentrations, the ratio of extracellular to intracellular volume is more favourable;
therefore, the negative effects of extracellular medium in the sample are less critical.
Whole cell broth extraction
Prepare 4 mL extraction solution in a centrifuge tube (see
Section 3
).
Spray 1 mL of culture broth into the extraction solution. If possible use the
bottom valve of the bioreactor or a rapid sampling device.
The extraction solution will arrest any metabolic activity and release metabolites
from the cells. At this point, extracellular metabolites can no longer be
distinguished from intracellular metabolites.
After extraction, centrifuge for 1 min at 12,000
g
at 4
C and store the
80
C.
supernatant at
Sampling of metabolites in the supernatant (optional)
Prepare 4 mL extraction solution in a centrifuge tube (see
Section 3
).
Transfer 2 mL of the culture broth into an Eppendorf-type tube and centrifuge
at 4
C.
