Biology Reference
In-Depth Information
Transfer 1 mL of the supernatant into the extraction solution. The extraction
solution will inactivate enzymes present in the culture supernatant.
Centrifuge the extract for 1 min at 12,000
g
and 4
C and store the supernatant
80
C.
at
3.3
High-throughput sampling of bacterial cultures in 96-well format
Recently, rapid sampling was adapted to a 96-well format by using fritted plates for
cultivation, which ensure fast sample transfer into cold methanol for quenching
(
Ewald
et al.
, 2009
). We adopted this method for bacteria using the whole cell broth
extraction technique adapted for the 96-well format.
Cultivate bacteria in a 96 deep well plate with fritted bottoms (Nunc, USA, no.
278011) using 500-600
m
L medium and monitor growth by withdrawal of
L samples.
Determine OD
600
just before sampling. All wells should be in a range of OD
600
0.5-1 for metabolome analysis.
Fill each well of a 48-square deep well plate with 3.6 mL extraction solution and
place it in a vacuum manifold.
Apply a moderate vacuum of
20-25
m
50 mbar.
Place the culture in the 96-well deep well plate with fritted bottoms on top of the
vacuum manifold and transfer the culture to the extraction solution in the 48-well
plate. Note that in this step two wells of the 96-well plate with
500
m
L are
pooled into a single well for extraction.
Quickly remove the vacuum, take out the 48-well plate and add 100
m
L internal
standard (see
Section 5.3
) to each well using a multi-channel pipette.
Close the 48-well plate with a foil or rubber seal and incubate for 5 min at 80
C
in a water bath (use glass beads and manual shaking to increase extraction
efficiency).
After incubation, immediately cool the plate to
4
C.
Centrifuge the 48-well plate 10 min at 5000 rpm and 4
C.
Transfer the supernatant into a new 48-well plate and dry the extracts under
vacuum at 0.12 mbar to complete dryness. Store the dry metabolite extracts at
80
C until required.
Re-suspend the dry metabolite extracts in 100
m
L water using a multi-channel
pipette and transfer into a 96-well storage plate.
Centrifuge the storage plate 10 min at 5000 rpm and 4
C and transfer 10-20
m
L
of the supernatant into another 96-storage plate which is then placed into the
autosampler for analysis using a mass spectrometry method (optional: transfer
supernatant into conical vials).
Using the above-described method will usually result in contamination of the sample
with salts from the culture medium and with extracellular metabolites, both of which
will interfere with the analysis. For specific questions, it might be applicable to wash
