Biology Reference
In-Depth Information
2.2
Inoculum
Plate the tested strain on Luria Bertani (LB) agar plates.
Transfer a colony to 5 mL of LB-medium in a 15-mL tube for pre-culturing and
cultivate for 3-5 h until the culture has an OD
600
of 0.5-1.
Transfer 5
m
L of the LB pre-culture to 5 mL of M9 medium in a 15-mL tube for a
second pre-culturing step. Since pre-culturing is usually done overnight, it is
convenient to prepare serial dilutions of the LB pre-culture to achieve an
appropriate OD the next day.
Add 35 mL of M9 medium to a 500-mL non-baffled shake flask and preheat for
15 min in an incubator at the desired temperature.
Inoculate the shake flask with the M9 pre-culture. Ideally, the pre-culture should
be in exponential growth phase with an OD
600
value between 0.5 and 1.0.
Determine the amount of pre-culture that has to be added for a starting OD
600
of 0.05 in the 35 mL M9 medium of the shake flask. Pellet the cells in the
required amount of pre-culture by centrifugation (3 min at 5000
g
,37
C).
Discard the supernatant and re-suspend the cell pellet in 100
m
L of fresh
medium.
Inoculate the shake flask with the re-suspended cells.
2.3
Cultivation
Check the OD of the culture following inoculation by removing 1 mL and
measuring the OD
600
.
Incubate the shake flask at the desired temperature on an orbital shaker with 5 cm
shaking diameter at 300 rpm.
Samples of supernatant (optional): after measuring the OD
600
, transfer the
undiluted sample into an Eppendorf-type tube and pellet the cells at 12,000
g
for 3 min. Decant the supernatant and store it in a separate Eppendorf tube at
20
C until required for the measurement of the concentrations of extracellular
substrate(s) and by-products.
Monitor the growth of the culture by sampling 1 mL of culture every hour and
measuring the OD
600
. Use appropriate dilutions in 9 g L
1
NaCl to ensure that the
measured OD
600
is in the linear range of the spectrophotometer (typically 0.02-
0.3 OD units).
Verify that the cells are growing exponentially (OD
600
<
1.5) when taking
samples for metabolome analysis. However, sampling at OD
600
<
0.5 reduces the
sensitivity of the analysis. The experiments should be performed in biological
replicates, usually two to three independent shake flask cultivations. The
reproducibility of physiological parameters, such as growth rate, substrate uptake
rate and by-product formation, is a measure of stable and robust cultivation
conditions. Reproducible physiology is a prerequisite for metabolomics
experiments since they are highly sensitive to these parameters.